Controlling protein levels in eucaryotic organisms

ABSTRACT

The invention relates to novel compounds comprising a ubiquitination recognition element and a protein binding element. The invention also relates to the use of said compounds for modulating the level and/or activity of a target protein. The compounds are useful for the treatment of disease such as infections, inflammatory conditions, cancer and genetic diseases. The compounds are also useful as insecticides and herbicides.

This application claims the benefit of U.S. Provisional Application No.60/119,851, filed Feb. 12, 1999, the entire content of which is herebyincorporated by reference in this application.

This is a divisional of application Ser. No. 09/406,781, filed Sep. 28,1999, now U.S. Pat. No. 6,306,663, the entire content of which is herebyincorporated by reference in this application.

FIELD OF INVENTION

The subject invention relates to novel compounds and their use incontrolling levels of proteins in eukaryotic organisms.

BACKGROUND OF INVENTION

Ubiquitin Mediated Protein Degradation

Ubiquitin is known to be one of several factors required forATP-dependent protein degradation in eukaryotic cells. One function ofintracellular protein degradation, most of which is ATP-dependent, isselective elimination of damaged and otherwise abnormal proteins.Another is to confer short half-lives on undamaged proteins whoseconcentrations in the cell must vary as functions of time, as is thecase, for example, with many regulatory proteins. Many other proteins,while long-lived as components of larger macromolecular complexes suchas ribosomes and oligomeric proteins, are metabolically unstable in afree, unassociated state. Ubiquitination is also involved in the controlof cell surface receptors such as platelet-derived growth factor (PDGF),the T cell receptor, G protein-coupled receptors and others. In additionto these proteins complexed with ubiquitin, ubiquitin is also foundcovalently linked to lipids in membranes (Guarino, L A, 1995, Cell 80,301-309).

Ubiquitin, a 76-residue protein, is present in eukaryotes either free orcovalently joined, through its carboxyl-terminal glycine residue, tovarious cytoplasmic, nuclear, and integral membrane proteins. A familyof ubiquitin-conjugating enzymes (also called E2 enzymes) catalyzes thecoupling of ubiquitin to such proteins (ubiquitination) generally incombination with a recognition element called E3 that may also functionto carry out the ubiquitination. The fact that the protein of ubiquitinis conserved among eukaryotes to an extent unparalleled among knownproteins suggests that ubiquitin mediates a basic cellular function.

It has been shown that selective degradation of many short-livedproteins requires a preliminary step of ubiquitin conjugation to atargeted proteolytic substrate. One role of ubiquitin is to serve as asignal for attack by proteases specific for ubiquitin-protein conjugates(Finley and Varshavsky, Trends Biochem. Sci. 10:343-348 (1985)).

At least some short-lived proteins are recognized as such because theycontain sequences (degradation signals) which make these proteinssubstrates of specific proteolytic pathways. The first degradationsignal to be understood in some detail comprises two distinctdeterminants: the protein's amino-terminal residue and a specificinternal lysine residue, the N-end rule (Bachmair et al., Science234:179-186 (1986); Bachmair and Varshavsky, Cell 56:1013-1032 (1989)).The N-end rule, a code that relates the protein's metabolic stability tothe identity of its amino-terminal residue (Bachmair et al., Science234:179-186 (1986), is universal in that different versions of the N-endrule operate in all of the eukaryotic organisms examined, from yeast tomammals (Gonda et al., J. Biol. Chem. 264:16700-16712 (1989)).

The second essential determinant of the N-end rule-based degradationsignal, referred to as the second determinant, is a specific internallysine residue in the substrate protein that serves as the site ofattachment of a multiubiquitin chain. Formation of the multiubiquitinchain on a targeted short-lived protein is essential for the protein'ssubsequent degradation. The enzymatic conjugation of ubiquitin to otherproteins involves formation of an isopeptide bond between thecarboxy-terminal glycine residue of ubiquitin and the epsilon-aminogroup of a lysine residue in an acceptor protein. In a multiubiquitinchain, ubiquitin itself serves as an acceptor, with several ubiquitinmoieties attached sequentially to an initial acceptor protein to form achain of branched ubiquitin—ubiquitin conjugates (Chau et al., Science243:1576-1583 (1989)).

The elucidation of the fundamental rules governing the metabolicstability of proteins in cells, and especially the deciphering of theN-end rule-based degradation signal, has made possible the manipulationof proteins to vary their half-lives in vivo (Bachmair and Varshavsky,Cell 56:1019-1032 (1989)).

The N-degron is an intracellular degradation signal whose essentialdeterminant is a specific (“destabilizing”) N-terminal amino acidresidue of a substrate protein. A set of N-degrons containing differentdestabilizing residues is manifested as the N-end rule, which relatesthe in vivo half-life of a protein to the identity of its N-terminalresidue. The fundamental principles of the N-end rule, and theproteolytic pathway that implements it, are well established in theliterature (see, e.g., Bachmair et al., Science 234: 179 (1986);Varshavsky, Cell 69: 725 (1992), U.S. Pat. Nos.: 5,122,463; 5,132,213;5,093,242 and 5,196,321) the disclosures of which are incorporatedherein by reference in their entirety.

In eukaryotes, the N-degron comprises at least two determinants: adestabilizing N-terminal residue and a specific internal lysine residue(or residues). The latter is the site of attachment of a multiubiquitinchain, whose formation is required for the degradation of at least someN-end rule substrates. Ubiquitin is a protein whose covalent conjugationto other proteins plays a role in a number of cellular processes,primarily through routes that involve protein degradation.

In a stochastic view of the N-degron, each internal lysine of a proteinbearing a destabilizing N-terminal residue can be assigned a probabilityof being utilized as a multiubiquitination site, depending ontime-averaged spatial location, orientation and mobility of the lysine.For some, and often for all of the Lys residues in a potential N-endrule substrate, this probability is infinitesimal because of thelysine's lack of mobility and/or its distance from a destabilizingN-terminal residue.

It is possible to construct a thermolabile protein bearing adestabilizing N-terminal residue in such a way that the protein becomesa substrate of the N-end rule pathway only at a temperature high enoughto result in at least partial unfolding of the protein. This unfoldingactivates a previously cryptic N-degron in the protein by increasingexposure of its (destabilizing) N-terminal residue, by increasingmobilities of its internal Lys residues, or because of both effects atonce. Since proteolysis by the N-end rule pathway is highly processive,any protein of interest can be made short-lived at a high(nonpermissive) but not at a low (permissive) temperature by expressingit as a fusion to the thus engineered thermolabile protein, with thelatter serving as a portable, heat-inducible N-degron module.

The heat-inducible N-degron module can be any protein or peptide bearinga destabilizing N-terminal residue that becomes a substrate of the N-endrule pathway only at a temperature high enough to be useful as anonpermissive temperature.

The idea of metabolically destabilizing a protein or peptide of interestusing a protein or peptide (ie targeting a protein or peptide fordegradation) has been described in U.S. Pat. No. 5,122,463. Thismetabolic destabilization requires that the protein or peptide ofinterest must contain a second determinant of the N-end rule-baseddegradation signal. The method comprises contacting the protein orpeptide of interest with a targeting protein or peptide that interactsspecifically with the protein or peptide of interest. The targetingpeptide or protein is characterized as having a destabilizingamino-terminal amino acid according to the N-end rule of proteindegradation.

The ability to activate the ubiquitination and degradation of otherproteins not containing an N-terminus N-degron signal has been shown ina multisubunit protein where the N-degron signals are located ondifferent subunits and still target a protein for destruction (U.S. Pat.No. 5,122,463). Moreover, in this case (trans recognition) only thesubunit that bears the second N-degron signal (lysine) determinant isactually degraded. Thus, an oligomeric protein can contain bothshort-lived and long-lived subunits. In these examples thedemonstrations are all based on known multisubunit proteins andalterations of these to bring about the destabilization of subunitsinvolved in these multisubunit complexes.

A different aspect of targeting the ubiquitination system based onchimeric proteins of E2 to achieve selective targeting and alterationsin the levels of proteins has been described (Gosink M M and Vierstra RD, 1995, Proc. Natl. Acad. Sci. 92, 9117-9121). These researchersdemonstrated that selective ubiquitination and degradation can beachieved using a protein, which is a fusion protein of a ubiquitinatingprotein with a binding protein.

In one interesting study of the N end rule, the degradation of DHFR wasstabilized by the binding of a small molecule indicating that bindingsmall molecules could prevent the degradation of proteins. This was alsosuggested in U.S. Pat. No. 5,122,463 where the idea of using peptidesand proteins to target the ubiquitination of proteins to which they bindis suggested. In this patent the peptides are described as binding insuch a way that the peptide interferes with the folding of the targetprotein “folding-interfering targeting peptides” suggesting also thatpeptides binding might prevent degradation as seen with DHFR. Indeed inthis patent the focus for the peptides is the sequence of the targetprotein to give rise to these destabilizing residues.

Other Protein Covalent Modification for Protein Targeting

A number of systems mirror the protein modification pathway ofubiquitin. Among these are based on the attachment of Apg12, Rub1/Nedd8and Smt3/SUMO-1 to proteins in addition to the ubiquitin pathway. Inthese systems homology at the level of sequence is seen but also clearparallels can be drawn based on the functional elements involved in thevarious systems (S Jentsch and H. D. Ulrich, Nature (1998) 395,321-322).

In the case of the Apg12 system this protein is involved in theautophagy of various cellular components. Apg12 appears to be thefunctional homologue of ubiquitin and is transferred via Apg7 and Apg10the functional homologue of the E1 and E2 ubiquitin conjugating enzymes,respectively. Apg12 transferred via Apg7 and Apg10 is used to modifyApg5 to activate autophagy. The analysis of the sequence of Apg7 shows aconsiderable homology to the E1 enzymes of the ubiquitin pathway. In thecase of Rub1/Nedd8 system this protein is involved in some regulatoryrole. The Smt3/SUMO-1 system is involved in the targeting of proteins.

Drug Targets

The number of drug targets for human therapeutics is around 400 humangene products, such as enzymes, receptors and ion channels. But theremay be 2500-5000 molecular targets whose exploitation may be capable ofrestoring function in the 100 or so common human polygenic diseases.Many of these new targets are being discovered by the intensive searchof the human genome by various groups using focused and random methods.

The following are examples of drug targets which are the subject ofinvestigation by various pharmaceutical companies: B7.1 and B7,TNFR1m(p55), TNFR2 (p75), NADPH oxidase, Bcl/Bax and other partners inthe apotosis pathway, C5a receptor, HMG-CoA reductase, PDE Vphosphodiesterase type, PDE IV phosphodiesterase type 4, PDE I, PDEII,PDEIII, squalene cyclase inhibitor, CXCR1, CXCR2, nitric oxide (NO)synthase, cyclo-oxygenase 1, cyclo-oxygenase 2, 5HT receptors, dopaminereceptors, G proteins ie Gq, histamine receptors, 5-lipoxygenase,tryptase serine protease, thymidylate synthase, purine nucleosidephosphorylase, GAPDH trypanosomal, glycogen phosphorylase, Carbonicanhydrase, chemokine receptors, JAK/STAT, RXR and similar, HIV 1protease, HIV 1 integrase, influenza, neuraminidase, hepatitis B reversetranscriptase, sodium channel, multi drug resistance (MDR), proteinP-glycoprotein (and MRP), tyrosine kinases, CD23, tyrosine kinase p56lck, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF-alphaR, ICAM1, Ca++channels, VCAM, VLA-4 integrin, selectins, CD40/CD40L, neurokinins andreceptors, inosine monophosphate dehydrogenase, p38 MAP Kinase,Ras/Raf/MEK/ERK pathway, interleukin-1 converting enzyme, caspase, HCV,NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotide formyltransferase, rhinovirus 3C protease, herpes simplex virus-1 (HSV-1),protease, cytomegalovirus (CMV) protease, poly (ADP-ribose) polymerase,cyclin dependent kinases, vascular endothelial growth factor, oxytocinreceptor, microsomal transfer protein inhibitor, bile acid transportinhibitor, 5 alpha reductase inhibitors, angiotensin II, glycinereceptor, noradrenaline reuptake receptor, endothelin receptors,neuropeptide Y and receptor, adenosine receptors, adenosine kinase andAMP deaminase, purinergic receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2X1-7),farnesyltransferases, geranylgeranyl transferase, TrkA a receptor forNGF, beta-amyloid, tyrosine kinase Flk-1/KDR, vitronectin receptor,integrin receptor, Her-2/neu, telomerase inhibition, cytosolicphospholipase A2, EGF receptor tyrosine kinase.

Insecticide target examples include, ecdysone 20-monooxygenase, ionchannel of the GABA gated chloride channel, acetylcholinesterase,voltage-sensitive sodium channel protein, calcium release channel, andchloride channels.

Herbicide target examples include Acetyl-CoA carboxylase,adenylosuccinate synthetase, protoporphyrinogen oxidase, andenolpyruvylshikimate-phosphate synthase.

These various targets are typically used in screens that look for acompound to alter the level of activity of the selected target andrequire the compound to be in solution. In some cases the assay todetermine activity in a potential compound has to be based on a cellbased assay. The best assays for compound screens are where theinteraction of two molecules is modulated allowing the development ofrapid assays based on the determination of binding.

In addition to the drawbacks of current drug and compound discoveryefforts described above, problems of specificity arise due to the commonbasis for the activity of various compounds. For example in trying tofind compounds which block the dopamine receptor, one is interested inthe inhibition of a specific receptor sub-type due to its expression ina selected tissue. The binding site of the receptor is designed to bindto dopamine and thus has a common structure across the various sub-typesof receptors. This homology of structure at the target site of thediscovery effort makes it difficult to identify compounds with optimallevels of specificity for given sub-types and thus difficult to achievethe levels of therapeutic affect desired.

The present invention provides a solution to this problem.

Antigen Presentation

The target degradation of various proteins in the cell is a mechanismfor the presentation of various peptides in the context of MHC. It hasbeen demonstrated that the ubiquitination of intracellular proteinsleads to the degradation of the protein via the 26S proteasome andenhanced presentation of the resultant peptides in the context of MHC I.This enhanced presentation leads to improved immune responses by thestimulation of various cells involved in the immune system. In manydiseases the antigenicity of various proteins does not appear to bepotent enough to generate a robust immune response. For example in thecase of cancer certain antigens are present but fail to elicite a potentimmune response (Tobery T and Siliciano R F., 1999, J Immunol. 162,639-642). The present invention provides a solution to this problem ofgenerating an improved immune response.

Antisense

Antisense technology is a novel drug therapy approach. Antisense drugswork at the genetic level to interrupt the process by which diseasecausing proteins are produced. Proteins play a central role in virtuallyevery aspect of human metabolism. Many human diseases are the result ofinappropriate protein production. Antisense drugs are designed toinhibit the production of disease causing proteins. These antisensedrugs function by binding to specific nucleic acid sequences in a celland block the production of specific proteins in this way a specificproteins level is reduced. Examples of targets for this technology arevirus-based diseases, cancer, Crohn's disease, renal transplantrejection, psoriasis, ulcerative colitis, and inflammation. The specifictargets are; HPV, HIV, CMV, hepatitis C, ICAM-1, PKC-alpha, c-rafkinase, Ha-ras, TNF-alpha and VLA-4.

SUMMARY OF INVENTION

The invention comprises compositions and methods for controlling thelevels of proteins in eukaryotic organisms. This control of proteinlevels is achieved using an exogenous molecule able to affectubiquitination of a given protein. The ubiquitinated protein is targetedfor intracellular degradation via normal cellular pathways. Theexogenous molecule able to selectively target ubiquitination of apre-selected protein comprise; a ubiquitination recognition element andtarget protein binding element for a pre-selected protein covalentlylinked to form the compositions of the invention.

The ubiquitination recognition element is designed to interact with theubiquitination mechanisms of the cell allowing their recruitment. Thetarget protein binding element binds to pre-selected protein in order toeffectively present the ubiquitin recognition element.

This invention offers a number of improvements over the art especiallyfor drug development. The invention provides a more cost effective routefor drug development and drugs with improved activity.

The invention comprises compounds for activating the ubiquitination of atarget protein comprising, a ubiquitination recognition element which isable to bind to either the E3 or E2 functional elements of theubiquitination system, the ubiquitination recognition element has amolecular weight less than 30,000 and has a binding affinity for the E3and/or E2 elements of the ubiquitination system of at least 10² M⁻¹ and;a target protein binding element that is able to bind specifically to atarget protein, the target protein binding element has a molecularweight of less than 30,000 and has a binding affinity for the targetprotein greater than 10⁵ M⁻¹, the ubiquitination recognition element iscovalently linked to the target protein binding element.

The invention also comprises compounds for activating the ubiquitinationof a target protein comprising, a ubiquitination recognition peptideelement which is able to bind to either the E3 or E2 functional elementsof the ubiquitination system, the ubiquitination recognition peptideelement has a molecular weight less than 30,000 and has a bindingaffinity for the E3 and/or E2 elements of the ubiquitination system ofat least 10² M⁻¹ and a target protein binding element that is able tobind specifically to a target protein, the target protein bindingelement has a molecular weight of less than 30,000 and has a bindingaffinity for the target protein greater than 10⁵ M⁻¹, the ubiquitinationrecognition peptide element is covalently linked to the target proteinbinding element.

The invention also comprises compounds for activating the ubiquitinationof a target protein comprising, a ubiquitination recognition elementwhich is able to bind to either the E3 or E2 functional elements of theubiquitination system, the ubiquitination recognition element has amolecular weight less than 30,000 and has a binding affinity for the E3and/or E2 elements of the ubiquitination system of at least 10² M⁻¹ anda target protein binding peptide element that is able to bindspecifically to a target protein wherein the target protein peptidebinding element has a molecular weight of less than 30,000 and has abinding affinity for the target protein greater than 10⁵ M⁻¹, whereinthe ubiquitination recognition element is covalently linked to thetarget protein binding peptide element.

The invention comprises compounds for activating the ubiquitination of atarget protein comprising, a ubiquitination recognition peptide elementwhich is able to bind to either the E3 or E2 functional elements of theubiquitination system, wherein the ubiquitination recognition peptideelement has a molecular weight less than 30,000 and has a bindingaffinity for the E3 and/or E2 elements of the ubiquitination system ofat least 10² M⁻¹ and a target protein binding peptide element that isable to bind specifically to a target protein wherein the target proteinbinding peptide element has a molecular weight of less than 30,000 andhas a binding affinity for the target protein greater than 10⁵ M⁻¹ wherethe ubiquitination recognition peptide element is covalently linked tothe target protein binding peptide element.

The invention also provides a method of modulating the level and/oractivity of at least one target protein in an eukaryotic cell via themodulation of ubiquitination of the at least one target proteincomprising contacting the cell with a compound comprising; aubiquitination recognition element which is able to bind to either theE3 or E2 elements of the ubiquitination system, wherein theubiquitination recognition element has a molecular weight less than30,000 and has a binding affinity for the E3 and/or E2 elements of theubiquitination system of at least 10² M⁻¹ and; a target protein bindingelement that is able to bind specifically to a target protein whereinthe target protein binding element has a molecular weight of less than30,000 and has a binding affinity for the target protein greater than10⁵ M⁻¹, the ubiquitination recognition element is covalently linked tothe target protein binding element.

The invention also provides a method of treating an infection in amammal comprising administering to the mammal an amount of a compoundsufficient to eliminate and/or reduce the infection comprisingcontacting the mammal with a compound comprising; a ubiquitinationrecognition element which is able to bind to either the E3 or E2elements of the ubiquitination system, wherein the ubiquitinationrecognition element has a molecular weight less than 30,000 and has abinding affinity for the E3 and/or E2 elements of the ubiquitinationsystem of at least 10² M⁻¹ and; a target protein binding element that isable to bind specifically to a target protein wherein the target proteinbinding element has a molecular weight of less than 30,000 and has abinding affinity for the target protein greater than 10⁵ M⁻¹, whereinthe ubiquitination recognition element is covalently linked to thetarget protein binding element.

The invention is also a method of treating cancer or tumor in a mammalcomprising administering to the mammal an amount of a compoundsufficient to reduce the size of the tumor comprising contacting themammal with a compound comprising; a ubiquitination recognition elementwhich is able to bind to either the E3 or E2 elements of theubiquitination system, wherein the ubiquitination recognition elementhas a molecular weight less than 30,000 and has a binding affinity forthe E3 and/or E2 elements of the ubiquitination system of at least 10²M⁻¹ and; a target protein binding element that is able to bindspecifically to a target protein wherein the target protein bindingelement has a molecular weight of less than 30,000 and has a bindingaffinity for the target protein greater than 10⁵ M⁻¹, wherein theubiquitination recognition element is covalently linked to the targetprotein binding element.

The invention also provides a method of generating a compound whichcomprises covalently linking a target protein binding element to aubiquitination recognition element.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the basic elements of the invention, where a moleculecontaining a ubiquitination recognition element and a target proteinrecognition element brings together the target protein and aubiquitination system to stimulate the ubiquitination of the targetprotein by the ubiquitination system.

FIG. 2 shows the synthetic steps for synthesis of L-chicoric acid.

FIG. 3 shows the synthetic steps for synthesis of N-bromoacetylethylenediamine

FIG. 4 shows the synthetic steps for synthesis of bromoacetylatedL-chicoric acid.

FIG. 5 shows the conjugation of the ubiquitination recognition elementto L-chicoric acid.

FIG. 6 shows the synthetic steps for synthesis of ubiquitinationrecognition element linked to glutathione.

FIG. 7 shows the synthetic steps for synthesis of ubiquitinationrecognition element linked to fluorescein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of development of compoundswhich are active via a new mechanism of action. The invention is to anew class of molecules that make use of the targeted modification and/ordegradation of proteins to modulate a selected target protein'sconcentration and/or activity. This is achieved through the constructionof a bi-functional molecule. This control of protein levels is achievedusing an exogenous molecule able to affect ubiquitination of a givenprotein. The ubiquitinated protein is targeted for intracellulardegradation via normal cellular pathways.

The exogenous molecules able to selectively target ubiquitination of apre-selected protein comprise; a ubiquitination recognition element andtarget protein binding element for a pre-selected protein covalentlylinked to form the compositions of the invention (FIG. 1).

The ubiquitination recognition element is designed to interact with theubiquitination mechanisms of the cell allowing their recruitment. Thetarget protein binding element binds to a pre-selected protein in orderto effectively present the ubiquitin recognition element.

Definitions

Ubiquitin, as used herein is a protein which is functionally andstructurally related to cellular ubiquitin. The functionally activity isdefined via its conjugation to other proteins forming covalent proteinconjugates through the action of an ATP dependent cellular pathway andits protein sequence. The structural relation is defined either bysequence homology and/or structure homology. Sequence homology isdefined by a BLAST sequence homology analysis (Altschul S F et al., JMol Biol 1990, 215, 403-410) where the E value is less than 0.063,representing a significant homology. Structural homology is defined by aVAST homology analysis where the p-value is less than 0.0001,representing a significant homology.

Ubiquitination, as used herein is the formation of a covalent bondbetween a cellular protein and a ubiquitin protein (as defined above)through the action of an ATP dependent cellular pathway.

Ubiquitination system, as used herein is a cellular system able todirect the formation of covalent protein conjugates between ubiquitinand other proteins. Ubiquitination systems consist of one or a number ofproteins involved in the activation of ubiquitin, recognition of aprotein for ubiquitination and formation of ubiquitin:proteinconjugates.

Ubiquitination recognition element, as used herein is a chemical moietywhich is able to bind with a ubiquitination systems proteins or itscomponent proteins. This binding is further defined by the ability ofthe chemical moiety to promote the ubiquitination of a protein attacheddirectly or indirectly to the moiety.

Ubiquitination recognition peptide element, as used herein is a peptidemoiety which is able to bind with a ubiquitination systems proteins(other than those of the N-end rule) or its component proteins. Thisbinding is further defined by the ability of the peptide moiety topromote the ubiquitination of a protein attached directly or indirectlyto the moiety.

Ubiquitination recognition site, as used herein is a sequence of aprotein which is known to act as the recognition site for ubiquitinationsystems. This ubiquitination recognition site is further defined by theability of the site to promote the ubiquitination of a protein attacheddirectly or indirectly to the site.

Target protein, as used herein is a protein selected for ubiquitinationusing a compound of the subject invention.

Target protein binding element, as used herein is a chemical moietywhich is able to bind to a target protein. Examples of these bindingelements include drugs and toxin molecules.

Target protein binding peptide element, as used herein is a peptidestructure which is selected to bind to a target protein.

The means by which the compositions of the invention are identified andsynthesized is described below.

The invention solves problems of library construction and screening bymaking use of the binding activity of a small molecule to developbiologically active and valuable molecules. This removes the problemsassociated with the synthesis of chemical libraries in that a) thecompounds can be screened bound to solid phase (in fact an advantage ofthe subject invention) and b) the presence of a linker element is autility of the subject invention which is commonly a problem in solidphase chemistries. These specific advantages in combination allow for anoptimal route to the generation of chemical libraries and theirscreening. These two elements combine synergistically resulting in rapiddrug development. In addition to these advantages, the hit rates foractive compounds is increased as generalized binding is optimal, notjust binding to the active site which limits the potential drugcompounds which may be found following conventional drug screeningapproaches. The invention also allows for the development of smallmolecule drugs whose development is problematic using traditionalmethods, for example finding a small molecule which can block theinteraction of two large proteins such as is seen with cell cellinteractions, some receptor ligand interactions, and intra cellularsignaling pathways.

Since the method of the subject invention does not make use of the‘active’ site of a given target protein, it is able to achieve a levelof specificity for a drug molecule previously considered extremelydifficult and uncertain using conventional drug discovery efforts. Thisadvantage stems from the constraints placed on existing drug discoveryefforts that are based on the need to inhibit an enzyme or receptorbinding site that is common to a series of different proteins indifferent tissues and with very different roles in the physiology of theorganism. These constraints are based on the common structural elementsin the binding or catalytic sites of these related proteins which formthe site for conventional drug discovery. The common structural elementstypically result in the selection of drugs that will inhibit the wholeseries of different proteins as these structural elements form the basisfor the binding of the drug molecules selected from the screen. Thusconventional drug screening approaches result in the selection of drughits which do not provide the degree of selectivity desired to bringabout a desired therapeutic affect. In the subject invention, since theactive site does not need to be the target for the selection ofmolecules that form the basis of the drug molecule, a significantimprovement in the discovery of highly selective drugs is achieved. Theconsequence is the development of drugs with an enhanced therapeuticvalue. This advantage is further enhanced by the ability of this drugdiscovery approach to make use of the whole surface of the given proteintarget to find molecules with the desired binding specificity. Thisadvantage is then combined with the ability to make use of a rapidscreen that is wholly based on the use of binding and thus achieves alevel of speed and through put not possible with other methods. Thisadvantage is of great value when the desire is to find a very specificinhibitor of a given member of a protein family that is highlyhomologous and thus extremely difficult or impossible for drug discoverybased on the effector, receptor or catalytic site of the given protein.This invention thus provides a means for the development of compounds ofthe invention which are variously; therapeutics, have variouspharmacological activities, herbicides, pesticides, insecticides,antivirals, antifungals, anti-parasitics and are able to selectivelymodify the performance of an organism.

The subject invention also includes a method to enhance theimmunogenicity of a given protein. This is achieved by enhancing thedegradation of a given protein via the 26S proteasome by selectiveubiquitination resulting in increased presentation of the antigen aspeptides in the context of MHC I. The ability to enhance theimmunogenicity of a given protein has great value in the treatment ofinfectious diseases (HIV, HBV, HCV, Herpes, etc) and also in thetreatment of cancer where the cancer antigen is not very immunogenic.The use of this approach for cancer treatment in combination with cancervaccine approaches and/or use of cytokines such as gamma interferon, isalso contemplated.

The subject invention also provides a method whereby a small molecule isused to regulate the levels of a protein genetically engineered into acell line or organism. This is achieved via the modification of a geneencoding the protein of interest to contain, in addition to the desiredactivity of the protein, a binding site for a small molecule able toactivate targeted covalent modification. This modified nucleic acidencoding an protein is then used to generate a genetically engineeredcell or organism. This approach allows for the specific modulation of agiven proteins action after the production of a genetically modifiedcell or organism on addition of compounds of the invention able toactivate targeted covalent modification.

The subject invention also permits the development of specific compoundsof the invention which can be used to target specific proteins fordegradation to allow the determination of a given proteins role withinthe cell or organism. This approach is useful in target validation forthe development of pharmaceuticals, for conducting basic research andfor target validation for may other discovery efforts directed to thediscovery of molecules able to bring about modulation of an amino acidslevels and/or function.

Target Protein Binding Elements

The target protein binding elements of the invention are molecularstructures which bind target proteins, and are used in the compounds ofthe invention to target the ubiquitination recognition elements to thetarget protein. These target protein binding elements are covalentlylinked to the ubiquitination recognition elements to form the compoundsof the invention and provide the linkage between these two elements ofthe compounds of the invention. When the target protein binding elementof a compound of the invention binds to a given target protein itpresents the ubiquitination recognition element to allow the activationof the ubiquitination pathway and subsequent ubiquitination of thetarget protein bound by the target protein binding element.

Target protein binding elements are small organic molecules defined bybinding to a predetermined target molecule, having a molecular weightfrom 50 to 30,000 and with a binding affinity of greater than 10⁵ M⁻¹for the target protein of interest. The binding affinity in anadvantageous embodiment is greater than 10⁶ M⁻¹. The molecular weight inan advantageous embodiment is between 50 and 3,000. The binding affinityin a more advantageous embodiment is greater than 10⁸ M⁻¹. The molecularweight in a more advantageous embodiment is between 100 and 2,000. Mostdrugs are typically either neutral, weak acids or bases. Examples ofknown specific drugs are phenytoin (pKa of 8.3) and aspirin (pKa of3.0).

Also target protein binding elements can be selected based on having atleast one the following characteristics; less than 50 H-bond donors, MWless than 5,000, ClogP or MLogP (calculated log P, based on the PomonaCollege Medicinal Chemistry program ClogP or using Molecular DesignLimited MACCS and ISIS based programs MlogP, logP (the logarithm of theoctanol/water partition coefficient) less than 6, sum of N's and O's (arough measure of H-bond acceptors) less 100.

Also target protein binding elements can be selected based having on atleast one the following characteristics; less than 5 H-bond donors, MWless than 500, ClogP or MLogP less than 5, sum of N's and O's (a roughmeasure of H-bond acceptors) less 10.

Also target protein binding elements can be selected based on having twoor more combinations of the following characteristics; less than 5H-bond donors, MW less than 500, ClogP or MLogP less than 5, sum of N'sand O's (a rough measure of H-bond acceptors) less 10 (Lipinski C A,1997, Adv. Drug Delivery Rev. 23, 3-25).

These target protein binding elements are different from peptides,proteins and DNA and RNA in that they are not highly charged or polar,are readily absorbed into the body due to the size and hydrophobicity.Also one of the other key properties of target protein binding elementsis the stability relative to proteins which are stable within narrowranges of temperature, pH and ionic strength due to the need to maintaina give structural conformation of the folded polypeptide chain. Peptidesalthough not as sensitive to the physical properties of an environmentare relatively unsuitable as drugs due to the poor biological stability,short half-life and poor bioavailability within cells and are notconsidered compounds of the invention.

Some examples of molecules which have moieties desired in a targetprotein binding element include drug molecules and molecules selectedfor binding and/or inhibition of various proteins functions, forexample; fluorescein, biotin, antigens, L-deprenyl, Omeprazole,Clavulanate, organoarsenical compounds such as 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein, p-aminophenylarsine oxide,p-aminophenylarsine oxide, chicoric acid, captopril, enalapril,lovastatin, proscar, indinivar, zileuton, L-372,460 (J. Med Chem 41,401, 1998), apomorphine, N-n-propylnorapomorphine, dihydrexidine,quinpirole, clozapine, haloperidol, nitrocaramiphen, and iodocaramiphen.

It is evident from the small sample above that numerous examples existsof chemistries which could form the basis of chemistries for targetprotein binding element. Also the numerous nature of these potentialtarget protein binding elements is illustrative of the potential easewith which such moieties can be discovered using routineexperimentation.

Compounds of the invention include small molecules used in veterinary,agricultural, food and environmental applications where a biologicaleffect is generated. Examples of compounds of the invention arefungicides, herbicides, pesticides, algaecides, insecticides,anti-virals, anti-parasitics etc. In addition compounds of the inventionare also molecules able to form covalent bonds with the target proteinsof interest, such as suicide inhibitors. Examples of well know drugsable to from covalent bonds, are as follows; L-deprenyl (Gerlach, M etal 1992, Eur. J. Pharmacol. 226, 97-108), Omeprazole (Howden, C W. 1991,Clin. Pharmacokinet, 20, 38-49) and Clavulanate (Neu, HC. 1990, J. Am.Acad. Dermatol, 22, 896-904). In addition to these well known moleculesare a considerable number of other small molecules known to formcovalent bonds specifically with various proteins. Also consideredcompounds of the invention are enzyme substrates that are used tocovalently modify proteins (such as farnesylation, phosphorylation,glycosylation, and gerenylation), where the natural enzyme substrate ismodified in such a way that it contains a ubiquitination recognitionelement.

Target Protein Binding Peptide Elements

The target protein binding peptide elements of the invention are peptidestructures which are selected to bind to target proteins and are used inthe compounds of the invention to target the ubiquitination recognitionelements, excluding those based on the N-end rule, to the targetprotein. These target protein binding peptide elements are covalentlylinked to the ubiquitination recognition elements, excluding those basedon the N-end rule to form the compounds of the invention and provide thelinkage between these two elements of the compounds of the invention.When the target protein binding peptide element of a compound of theinvention binds to a given target protein it presents the ubiquitinationrecognition element to allow the activation of a ubiquitination pathway(not based on the N-end rule) and subsequent ubiquitination of thetarget protein bound by the target protein binding peptide element.Examples of these are peptide selected from combinatorial libraries suchas those expressed on the surface of phage (Yanofsky S D et al., Proc.Natl. Acad. Sci USA 1996, 93, 7381). Examples of target protein bindingpeptide elements include;

epsilon-aminocaproic acid-phospho-Y-E-E-I (SEQ ID #56) binding to srcSH2 domain;

DREGCRRGWVGQCKAWFN (SEQ ID #57) binding to erythropoietin;

ETPTFTWEESNAYYWQPYALPL (SEQ ID #58) binding to IL-1alpha;

TFVYWQPYALPL (SEQ ID #59) binding to IL-1alpha;

VSLARRPLPPLPGGK (SEQ ID #60) binding to the SH3 domains of Src, Fyn,Lyn, Yes, PI3K;

KGGGAAPPLPPRNRPRL (SEQ ID #61) binding to the SH3 domains of Src, Fyn,Lyn, Yes;

AECHPQGPPCIEGRK (SEQ ID #62) binding to streptavidin;

GACRRETAWACGA (SEQ ID #63) binding to alpha5beta1 integrin;

DITWDQLWDLMK (SEQ ID #64) binding to E-selectin;

RNMSWLELWEHMK (SEQ ID #65) binding to E-selectin;

Targets of the Target Protein Binding Element

Targets of the target protein-binding element are numerous and areselected from proteins and proteins that are expressed in a cell suchthat at least a portion of the sequences is available within the cell.The term protein includes all sequences of amino acids greater than twoand includes peptides. Below is a partial list of target proteins. Anyprotein in eukaryotic cells are targets for ubiquitination mediated bythe compounds of the invention. Those of special interest are thosewhich are involved in diseases or disease processes included; areinfectious diseases of viral, microbial, and parasitic nature, metabolicdiseases, aging, environmental diseases, genetic diseases, life stylediseases. Also protein targets which are involved in performanceenhancement are also targets, such as those involved in growth anddevelopment, memory, and sensory perception.

Examples of viruses contemplated as targets of the subject invention areHIV1, HIV2, HLTV, CMV, HPV, HSV, hepatitis, HBV, HCV, HAV, HDV, HGV,influenza A, influenza B, influenza C, rhinoviruses, rotaviruses,entroviruses, Ebola, polio, chicken pox, RSV, coronavirus, adenoviruses,parainfluenza 3, coxsackie A, and epstein-barr virus.

The following are example of targets of the target protein bindingelements of the subject invention, which include:

Receptors

CD124, B7.1 and B7, TNFR1m(p55), TNFR2 (p75), Bcl/Bax and other partnersin the apotosis pathway, C5a receptor, CXCR1, CXCR2, 5HT receptors,dopamine receptors, G proteins, ie Gq, histamine receptors, chemokinereceptors, JAK/STAT cf ligand, RXR and similar, CD4, CD5, IL-2 receptor,IL-1 receptor, TNF-alphaR, ICAM1, VCAM, VLA-4 integrin, selectins,CD40/CD40L, neurokinins and receptors, Ras/Raf/MEK/ERK pathway, vascularendothelial growth factor, oxytocin receptor, microsomal transferprotein inhibitor, angiotensin II, glycine receptor, noradrenalinereuptake receptor, endothelin receptors, neuropeptide Y and receptor,adenosine receptors, purinergic receptors (P2Y1, P2Y2, P2Y4, P2Y6,P2X1-7), TrkA a receptor for NGF, beta-amyloid, tyrosine kinaseFlk-l/KDR, vitronectin receptor, integrin receptor, Her-2/neu, MCHreceptor, IL-4 receptor alpha chain and the Toll-like receptors andhuman homologue, FKHR and AFX or the human homologues of daf2, daf16 andage1.

Enzymes

NADPH oxidase, HMG-CoA reductase, PDE V phosphodiesterase type, PDE IVphosphodiesterase type 4, PDE I, PDEII, PDEIII, squalene cyclaseinhibitor, nitric oxide (NO) synthase, cyclo-oxygenase 1,cyclo-oxygenase 2,5-lipoxygenase, tryptase serine protease, thymidylatesynthase, purine nucleoside phosphorylase, GAPDH trypanosomal, glycogenphosphorylase, Carbonic anhydrase, HIV 1 protease, HIV 1 integrase,influenza, neuraminidase, hepatitis B reverse transcriptase, tyrosinekinases, CD23, tyrosine kinase p56 lck, inosine monophosphatedehydrogenase, p38 MAP Kinase, interleukin-1 converting enzyme, caspase,HCV, NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotideformyl transferase, rhinovirus 3C protease, herpes simplex virus-1(HSV-1) protease, cytomegalovirus (CMV) protease, poly (ADP-ribose)polymerase, cyclin dependent kinases, 5 alpha reductase inhibitors,adenosine kinase and AMP deaminase, farnesyltransferases, geranylgeranyltransferase, telomerase, cytosolic phospholipase A2, EGF receptortyrosine kinase.

Membrane Transporters

Sodium channel, Ca⁺⁺ channels, multi drug resistance (MDR), proteinP-glycoprotein (and MRP), bile acid transporter.

Insecticide target examples include, ecdysone 20-monooxygenase, ionchannel of the GABA gated chloride channel, acetylcholinesterase,voltage-sensitive sodium channel protein, calcium release channel, andchloride channels.

Herbicide target examples include Acetyl-CoA carboxylase,adenylosuccinate synthetase, protoporphyrinogen oxidase, andenolpyruvylshikimate-phosphate synthase.

Targets for anti-parasitic drug development include: Leishmania,proteins of the sterol synthesis pathway: Plasmodium, dihydrofolatereductase; dihydrofolate reductase-thymidylate synthase (bifunctional)resistance known due to mutations in the gene for this enzyme, hemepolymerase: Trypanosoma, ornithine decarboxylase, trypanothionereductase, Ornithine decarboxylase of the trypanosoma represents anideal candidate for destruction due to its long half-life and low turnover in trypanosoma. It has also been suggested that the shikimatepathway, which is a target for herbicide development, would also be ofvalue in the development of anti-parasitics for parasites of the phylumApicomlexa (ie. Plasmodium falciparum, Cryptosporidium parvum andToxoplasma gondii) as it absent from mammals.

Also considered targets of the subject invention are proteins that areinvolved in the performance of a cell and/or organism. Performancecharacters of a cell and/or organism are those characters which areconsidered desirable traits which a cell and/or organism has in somepart. These performance characters may be present in other cells ororganisms and be desired in cells and/or organisms which do not possesthem. Examples of what are considered performance characters are, flowercolor, fragrances, specific shapes and colors in organisms such a catsand dogs, disease resistance, growth rates, size, taste, alcohol yieldfrom yeast. Thus performance characters are generally things that aredesired in cells and organisms used either in the production of desiredproducts or in the production of esthetical value (look, taste, feel,smell and sound).

In the case of flower color (considered an esthetical value), theproteins involved in the biosynthesis of flavonoids, carotenoids andanthocyanins; including flavanone 3 -hydroxylase, anthocyanin synthase,dihydroflavonol 4-reductase, flavonoid 3′,5′-hydroxylase, anthocyanin5-aromatic acyltransferase, UDP-glucose: flavonoid3-O-glucosyltransferase, anthocyanin rhamnosyltransferase, anthocyanin3′-methyltransferase, anthocyanin 3′5′-methyltransferase,leucoanthocyanidin dioxygenase, anthocyanidin synthase, anthocyaninacyltransferase, chalcone synthase, chalcone flavanone isomerase,glutathione S-transferase, one considered as targets of the subjectinvention involved in performance characteristics. In addition to theproteins involved in the synthesis of flower color, the proteinsinvolved in the regulation of the expression of the synthases and otherproteins involved in the production of flower color are also consideredtargets of the subject invention. Examples of the regulatory genesinclude the R and C1 gene families, an2 and jaf13, the delila gene.Quattrocchio F. 1998, Plant J. 13(4), 475-488.

Other potential target molecules of the subject invention includetargets as described above but also targets in all eukaryotic organisms.Potential targets exist in agriculture, veterinary and environmentalfields. For example in the agricultural field, molecules which areselective in action and non-toxic are highly desirable for use asherbicides, anti-virals, anti-parasitics, growth modulators and drugs;thus target molecules can be selected from certain animals, plants,viruses and parasites of interest to agriculture. In the veterinaryfield anti-virals, anti-parasitics, antibiotics, growth modulators,anti-inflammatory and drugs are of interest and target molecules can beselected from certain animals, viruses and parasites of interest inveterinary science. In the environmental field the potential targets arethe same as for agricultural but the aims are to control selectivelycertain populations either positively as in the case of an endangeredspecies but also negatively where a population has expanded itsenvironment or where a foreign organism is undesirable to a givenecosystem. Thus it is understood by those skilled in the art that theability to modulate the level of a selected target molecule could havewide ranging effects in very diverse areas of science, technology andhuman endeavors.

Ubiquitin

In this invention ubiquitin includes ubiquitin and ubiquitin likesequences related either by sequence homology, by structural homology orfunctional homology or having been described as related to ubiquitin inthe scientific literature. Functional homology to ubiquitin is definedbased on a proteins ability to be attached covalently to other proteinsvia an ATP dependent enzyme system, proteins transferred in this way areconsidered to be ubiquitin in this invention and the protein couplingstep is considered to be ubiquitination. In the case of sequencehomology protein sequences with a BLAST (Altschul SF et al., J Mol Biol1990, 215, 403-410) E (Expected) value of 0.063 or less are consideredin this invention to be ubiquitin. The BLAST search being run the NIHweb server NIH, http://www.ncbi.nlm.gov). The E value is a parameterthat describes the chances of finding a sequence match based on chance.Thus the smaller the value the smaller the chance that the matchoccurred by chance. In the case of structural homology these aredetermined based on the VAST (NIH, http://www.ncbi.nlm.gov) analysis toyield p-value of less than 0.0001 are considered to be ubiquitin in thisinvention. The VAST (NIH, http://www.ncbi.nlm.gov) p-value is a measureof the significance of a comparison, expressed as a probability. Forexample if the p-value is 0.0001 then the odds are 10,000 to 1 againstseeing a match by chance.

A number of systems are also considered to be ubiquitination pathways inthis invention as these protein modification pathways, involved in theattachment of Apg12, Rub1/Nedd8 and Smt3/SUMO-1 are generally consideredas being equivalent to the ubiquitin pathway due to their functionalhomology to the ubiquitination pathway.

In these systems homology at the level of sequence is seen but alsoclear parallels can be drawn based on the functional elements involvedin the various systems (S Jentsch and H. D. Ulrich, Nature (1998) 395,321-322).

In the case of the Apg12 system this protein is involved in theautophagy of various cellular components. Apg12 appears to be thefunctional homologue of ubiquitin and is transferred via Apg7 and Apg10the functional homologue of the E1 and E2 conjugating enzymes of theubiquitin and final is used to modify Apg5 to activate autophagypossible via a targeting mechanism. The analysis of the sequence of Apg7shows a considerable homology to the E1 enzymes of the ubiquitinpathway.

In the case of Rub1/Nedd8 system these proteins are involved in aregulatory role. The Smt3/SUMO-1 system is also involved in thetargeting of proteins.

Antigens

Antigens of this invention are considered to be target proteins of thesubject invention. Antigens of the invention are proteins that arederived from numerous sources, examples of which are intracellularproteins of the host, or other target organisms. The antigens can alsobe derived from other organisms and are presented intracellularly withinthe organism of interest. Typically these antigens are derived from aninfectious organism such as a virus, bacteria or fungi or derived from anormal protein or one mutated in a given disease tissue. In oneembodiment of the invention antigens present in cancer cells areutilized. Example of cancer antigens include MAGE 1, MAGE 2, MAGE 3,tyrosinase, tyrosinase related protein 1 and 2. Pmel H. In the case ofviruses examples of antigens are proteins for example from HCV, HIV,HPV, HBV, influenza. rhinoviruses.

Ubiquitination Recognition Elements

In order to develop the compounds of the invention for targetedubiquitination, identification of a chemical element able to replace thetargeting and/or signaling activity of the N-terminal amino acid of aprotein or a sequence element of a protein, which result in theubiquitination of the protein, is required. A number of chemicalentities ‘ubiquitination recognition elements’ have already beendescribed which interact with the ubiquitination system of the cell; anumber of di-peptides, and some modified amino acids. These compoundshave been described based on the ability to inhibit the activity of theubiquitination pathway based on the known activating amino acids fromthe N-end rule. These include dipeptides, amino acid hydroxamates, andamino acid methyl esters with small uncharged, basic or bulkyhydrophobic N-terminal residues (Gonda et al 1989, J Biol. Chem. 264:16700). In addition sequence elements have been defined which include,‘destruction box’ or D box, PEST motifs, Deg1, Deg 2, delta (δ) domains,and phosphorylated sequences which also target ubiquitination. Alsoconsidered as ubiquitination recognition elements are oxidizedderivatives of peptides. These compounds are useful in the presentinvention. Examples of oxidized amino acid are oxidized methionine toform methionine sulfoxide, oxidized leucine to form hydroxyleucine,oxidized tryptophan to form N-formyl-kynurenine, and oxidized tyrosineto form 3,4-dihydroxyphenylalanine.

It is also understood that identification of new ubiquitinationrecognition elements is possible using standard methods for drugdiscovery (as outlined below) based on modulation of the ubiquitinationpathways. Methods of screening compounds which can be used asubiquitination recognition elements has been demonstrated fordipeptides, amino acid hydroxamates, and amino acid methyl esters withsmall uncharged, basic or bulky hydrophobic N-terminal residues and alsolarge chemical libraries (U.S. Pat. No. 5,766,927; WO 98/23283; GB2,320,570 Gonda et al 1989, J Biol. Chem. 264: 16700). In one specificexample a compound was identified ‘compound of example 2’(1-chloro-2,4-bis{4-[2-chloro-6-(5-(2,7-disulfo-4-hydroxy-3-(2-(1-sulfonaphthyl)azo)naphthyl)amino)-1,3,5-triazinyl]amino}benzene),of patent application GB 2,320,570 (which is hereby incorporated byreference in its entirety), which inhibited an E2 ubiquitinationreaction indicating its utility as a ubiquitination recognition elementof the subject invention; here in called compound Z. Other equivalentmethods for identification of chemical elements equivalent to the PEST,Deg and other sequence elements based on the assays for ubiquitinationsystems (Hochstrasser M and Varshavsky A 1990, Cell 61; 697-708) can beused. In the case of the PEST sequence from ornithine decarboxylase(amino acids 422-462), this sequence has been fused to a greenfluorescent protein sequence to generate a fluorescent protein which hasa half life of only 2 hours from its original >24 hours making this anideal system in which to detect molecular equivalent of the PESTsequence. In addition to specific molecular species which interact withthe E3 elements of the ubiquitination pathway, it is also contemplatedthat specific molecular species that interact with the E2 elements ofthe ubiquitination pathway can be used in an equivalent way to targetselective ubiquitination. A specific example of an E2 domain that isinvolved in targeting specific ubiquitination is the C-terminal domainsof E2. Thus chemical elements able to bind to the E2's and especiallythe C-terminal domains of E2 are considered ubiquitination recognitionelements of the subject invention. The elements that interact with theE2 elements have been defined as various sequence elements (parts) ofproteins that control their ubiquitination.

Analysis of the E3s has determined common themes in structure andfunction. The basic function for E3s is the recognition of a proteinsubstrate for ubiquitination. This is achieved either as a singleprotein or as a multi-protein complex. In some cases the E3s are singleproteins which typically depend on E2 to mediate the ubiquitination. Inthe case of one class of E3s known as SCF complexes (containing Skp1p,Cdc53p and F-box proteins in a complex) it is known that the F-boxproteins act as the substrate specific adapters to recruit varioussubstrates to the complex for ubiquitination. Thus in these E3s it isthe interaction of the F-box proteins with the proteins targeted forubiquitination, the ubiquitination is achieved through Cdc-34p (E2). Theabove description of the ubiquitination elements has drawn on generalnames for the elements such as E1, E2 and E3 but also some specificnames of the proteins in a given system. It will be understood by thoseskilled in the art that equivalent proteins, as determined by functionand sequence homology exist and can be considered to be equivalent(Patton E E, et, al. Trends Genet, 1998 14, 236-243).

Thus it is clear to those skilled in the art that binding moleculeswhich bind to the ubiquitination recognition site (FIG. 1) of theubiquitination system can be selected and identified using art knownmethods and available chemical libraries. In the absence of availablechemical libraries, synthesis of equivalent chemical libraries can bedone following art known methods, as described below for the discoveryof ubiquitination recognition elements of the subject invention.

Examples of PEST sequences include,

MEFMHISPPEPESEEEEEHS (SEQ ID NO 1), MEFMHESHSS (SEQ ID NO 2),MEFMHISPPEPESHSS (SEQ ID NO 3), MEFMHESEEEEEHSS (SEQ ID NO 4),MEASEEEEEF (SEQ ID NO 5), HGFPPEVEEQDDGTLPMSCAQESGMDRH (SEQ ID NO 6),HGFPPAVAAQDDGTLPMSCAQESGMDRH (SEQ ID NO 7), HGFPPEVEEQDDGALPMSCAQESGMDRH(SEQ ID NO 8), HGFPPEVEEQDDGTLPMSCAQESGMDHH (SEQ ID NO 9),HGFPPEVEEQDVGTLPMSCAQESGMDRH (SEQ ID NO 10),HGFPPEVEEQDVGTLPISCAQESGMDRH (SEQ ID NO 11),HGFPPEVEEQDASTLPVSCAWESGMKRH (SEQ ID NO 12), FPPGVEEPDVGPLPVSCAWESGMKRH(SEQ ID NO 13), FLAEVEEQDVASLPLSCACESGIEYPA (SEQ ID NO 14),

expressed as a following consensus

FXXEVEEQDXXXLPXSCAXESGXX(X) (SEQ ID NO 15), FXXAVAAQDXXXLPXSCAXESGXX(X)X(SEQ ID NO 16), or HGXXPEVX(XX)DXXXLXXSCAQESGMXXX (SEQ ID NO 17),

where X is any amino acid and (X) is an optional amino acid.

RHALDDVSN (SEQ ID NO 18), RLALNNVTN (SEQ ID NO 19), RAALGDVSN (SEQ ID NO20), RQVLGDIGN (SEQ ID NO 21), RAALGDLQN (SEQ ID NO 22), RAALGNISN (SEQID NO 23), RNTLGDIGN (SEQ ID NO 24), RTALGDIGN (SEQ ID NO 25), RAALGEIGN(SEQ ID NO 26), RAVLEEIGN (SEQ ID NO 27), RSAFGDITN (SEQ ID NO 28),RSILGVIQS (SEQ ID NO 29), RAALGVITN (SEQ ID NO 30), RTVLGVIGDN (SEQ IDNO 31), RTVGVLQEN (SEQ ID NO 32), RAALGTVGE (SEQ ID NO 33), RTVLGVLTEN(SEQ ID NO 34), RAALAVLKSGN (SEQ ID NO 35), RLPLAAKDN (SEQ ID NO 36),RQLFPIPLN (SEQ ID NO 37), RRTLKVIQP (SEQ ID NO 38),

expressed as a general structure

R(A/T)(A)LGX(I/V)(G/T)(N) (SEQ ID NO 39), or expressed as a consensusRXXLGXIXN (SEQ ID NO 53), where X is any amino acid and amino acids inparentheses occur in more than 50% of known destruction sequences.

Examples of other ubiquitination recognition elements are;

KEFAVPNETSDSGFISGPQSS (cactus) (SEQ ID NO 40), KGPDEAEESQYDSGLESLRSLR(IkBepsilon) (SEQ ID NO 41), KAADADEWCDSGLGSLGPDA (IkBbeta), (SEQ ID NO42), KKERLLDDRHDSGLDSMKDEE (IkBalpha), (SEQ ID NO 43),

with a consensus of

KX(8-10)DSG(hydrophobic amino acid)XS (SEQ ID NO 44), where the S inbold are phosphorylated. In addition to the signals associated with NFkB activation are the related ubiquitination recognition elementsSYLDSGIHSGAT (SEQ ID NO 45), (human beta-catenin) and RAEDSGNESEGE (SEQID NO 46), (HIV-1 Vpu) where the S in bold are phosphorylated.

The identified and/or discovered chemical entities which bind to thesites on the E3 and/or E2 elements involved in recognition prior toubiquitination are the ubiquitination recognition elements of thesubject invention. The ubiquitination recognition elements are thusfunctionally defined by their ability to compete for binding of thenatural recognition signals for ubiquitination with their ubiquitinationpartners, have a molecular weight less than 30,000; 50 to 10,000; 50 and3,000; 100 and 3,000; 200 and 3,000, are capable of being linked toother molecular species and retain their ability to compete for bindingof the natural recognition signals for ubiquitination with theirubiquitination partners. In addition the binding affinity of theseubiquitination recognition elements is typically greater than 10² M⁻¹.The binding affinity in a advantageous embodiment is greater than 10³M⁻¹. The binding affinity the most advantageous embodiment is greaterthan 10⁴ M⁻¹.

Some examples of ubiquitination recognition elements based on theN-recognin include;

Arg-εAhx-Cys (SEQ ID NO:66) Arg-β-Ala-εAhx-Cys Arg-εAhx-εAhx-CysPhe-εAhx-Cys Phe-β-Ala-εAhx-Cys Phe-εAhx-εAhx-Cys Arg-Ala-εAhx-CysArg-Ala-β-Ala-εAhx-Cys Arg-Ala-εAhx-εAhx-Cys Phe-Ala-εAhx-Cys (SEQ IDNO:67) Phe-Ala-β-Ala-εAhx-Cys Phe-Ala-εAhx-εAhx-Cys

Ubiquitination Recognition Peptide Element

Ubiquitination recognition peptide element, is defined as a peptidebased moiety which is able to bind with a ubiquitination systemsproteins (other than those of the N-end rule) or its component proteins.This binding is further defined by the ability of the peptide moiety topromote the ubiquitination of a protein attached directly or indirectlyto the moiety.

Examples of such ubiquitination recognition peptide elements are;

MEFMHISPPEPESEEEEEHS (SEQ ID NO 1), MEFMHESHSS (SEQ ID NO 2),MEFMHISPPEPESHSS (SEQ ID NO 3), MEFMHESEEEEEHSS (SEQ ID NO 4),MEASEEEEEF (SEQ ID NO 5), HGFPPEVEEQDDGTLPMSCAQESGMDRH (SEQ ID NO 6),HGFPPAVAAQDDGTLPMSCAQESGMDRH (SEQ ID NO 7), HGFPPEVEEQDDGALPMSCAQESGMDRH(SEQ ID NO 8), HGFPPEVEEQDDGTLPMSCAQESGMDHH (SEQ ID NO 9),HGFPPEVEEQDVGTLPMSCAQESGMDRH (SEQ ID NO 10),HGFPPEVEEQDVGTLPISCAQESGMDRH (SEQ ID NO 11),HGFPPEVEEQDASTLPVSCAWESGMKRH (SEQ ID NO 12), FPPGVEEPDVGPLPVSCAWESGMKRH(SEQ ID NO 13), FLAEVEEQDVASLPLSCACESGIEYPA (SEQ ID NO 14),

!

expressed as a following consensus

FXXEVEEQDXXXLPXSCAXESGXX(X) (SEQ ID NO 15), FXXAVAAQDXXXLPXSCAXESGXX(X)X(SEQ ID NO 16), or HGXXPEVX(XX)DXXXLXXSCAQESGMXXX (SEQ ID NO 17),

where X is any amino acid and (X) is an optional amino acid.

Examples of D boxes include,

RHALDDVSN (SEQ ID NO 18), RLALNNVTN (SEQ ID NO 19), RAALGDVSN (SEQ ID NO20), RQVLGDIGN (SEQ ID NO 21), RAALGDLQN (SEQ ID NO 22), RAALGNISN (SEQID NO 23), RNTLGDIGN (SEQ ID NO 24), RTALGDIGN (SEQ ID NO 25), RAALGEIGN(SEQ ID NO 26), RAVLEEIGN (SEQ ID NO 27), RSAFGDITN (SEQ ID NO 28),RSILGVIQS (SEQ ID NO 29), RAALGVITN (SEQ ID NO 30), RTVLGVIGDN (SEQ IDNO 31), RTVGVLQEN (SEQ ID NO 32), RAALGTVGE (SEQ ID NO 33), RTVLGVLTEN(SEQ ID NO 34), RAALAVLKSGN (SEQ ID NO 35), RLPLAAKDN (SEQ ID NO 36),RQLFPIPLN (SEQ ID NO 37), RRTLKVIQP (SEQ ID NO 38),

expressed as a general structure

R(A/T)(A)LGX(I/V)(G/T)(N) (SEQ ID NO 39), or expressed as a consensusRXXLGXIXN (SEQ ID NO 53), where X is any amino acid and amino acids inparentheses occur in more than 50% of known destruction sequences.

Examples of other ubiquitination recognition elements are;

KEFAVPNETSDSGFISGPQSS (cactus) (SEQ ID NO 40), KGPDEAEESQYDSGLESLRSLR(IkBepsilon) (SEQ ID NO 41), KAADADEWCDSGLGSLGPDA (IkBbeta), (SEQ ID NO42), KKERLLDDRHDSGLDSMKDEE (IkBalpha), (SEQ ID NO 43),

with a consensus of

KX(8-10)DSG(hydrophobic amino acid)XS (SEQ ID NO 44), where the S inbold are phosphorylated. In addition to the signals associated with NFkB activation are the related ubiquitination recognition elementsSYLDSGIHSGAT (SEQ ID NO 45), (human beta-catenin) and RAEDSGNESEGE (SEQID NO 46), (HIV-1 Vpu) where the S in bold are phosphorylated.

Ubiquitination Recognition Signal

Ubiquitination recognition signal is a sequence of a protein which isknown to act as the signal for ubiquitination systems. Thisubiquitination recognition signal has the ability to promote theubiquitination of a protein attached directly or indirectly to thesignal.

Method for the Selection of the Target Protein Binding Elements

The subject invention provides a significant advantage over the existingart as it makes use of binding to develop drugs and other compounds withactivity against selected target proteins. This advance over thetraditional methods is that the invention obviates the need to find acompound which binds to a specific site, by making the whole proteinsurface available for the development of drugs and other biologicallyactive compounds. This approach thus provides a new avenue for thediscovery and selection of novel pharmaceuticals, drugs and othervaluable biologically active compounds.

It is understood by those skilled in the art that methods for thediscovery of target protein binding elements to a pre-selected (target)proteins (targets of the subject invention) are well know. Examples arereferenced as follows, Karet G. Drug Discovery and Development Jan 1999,32-38, www.rdmad.com/drug; Bohm, H-J and Klebe, G., 1996, Angew. Chem.Int. Ed. Engl. 35, 2588-2614; Angew Chem Int Ed Engl 1996, 35,2288-2337; Bunin B A., 1996, Methods in Enzymology, 267, 448-465; PatekM. 1995, Tetrahedron Let., 36, 2227-2230; Nestler H P., 1996, Bioorg.Med. Chem. Lett., 6(12), 1327-1330; Look, G C., 1996, Bioorg. Med. Chem.Lett., 6(6), 707-712; Nakayama GR., 1998, Curr. Opin. Drug Discovery andDevelopment 1(1), 85-91; Hill D C., 1998 Curr. Opin. Drug Discovery andDevelopment 1(1). 92-97; Bright, C., 1998, Bioorganic and Med. Chem.Lett. 8, 771-774; Forbes I T., 1998, J Med. Chem. 41(5), 655-657; whichare hereby incorporated by reference in their entirety.

The binding molecules of the subject invention are defined by binding tothe selected target molecule, having a molecular weight less than30,000; 50 to 10,000; 50 to 3,000; 50 to 1,000; 100 to 3,000; 200 to3,000 and 300 to 3,000. Also the binding molecule is defined by thebinding affinity which is typically greater than 10⁵ M⁻¹. The bindingaffinity in an advantageous embodiment is greater than 10⁶ M⁻¹. Thebinding affinity in a more advantageous embodiment is greater than 10⁷M⁻¹. The binding affinity in the most advantageous embodiment is greaterthan 10⁸ M⁻¹.

Large libraries of compounds exist in numerous places. These comprisecompounds isolated from various natural sources in addition to thosegenerated denovo or partially denovo from natural precursor organicmolecules. The sources for various compound libraries include: ArQule(www.arqule.com); Pharmacopeia (www.pharmacopiea); Cerep(www.cerep.com); Merk; Glaxo-Welcome; Zenova; Sigma-Aldrich; OxfordAsymmetry International (www.oai.co.uk); Specs and BioSpecs(www.specs.net); AsInEx (www.asinex.com); ComGenex, Princeton, N.J.;Panax, New York, N.Y.;

Synthetic Approaches for Compound Generation to Screen for TargetProtein Binding Elements and Ubiquitination Recognition Elements of theSubject Invention

Combinatorial chemistry has been widely adopted by large and small drugdiscovery companies alike since 1990. This is a set of techniques forcreating a multiplicity of compounds and then testing them for activity(Angew Chem Int Ed Engl 1996, 35, 2288-2337). Combinatorial chemistry isused to generate large libraries of molecules instead of synthesizingcompounds one by one, as has been done traditionally. These librariesare screened using high-throughput screening to identify the mostpromising pharmaceutical compounds. Typical rates for success are around0.1% and libraries of around 200,000 compounds are typically screened.These initial hits in screens are then further analyzed for otherdesired drug properties for example drug metabolism, bio-availability,stability, potency, and cost. Thus, the discovery of compounds withbinding and inhibitory activity is a routine practice. This isespecially true if only binding is screened for independent ofmodulation to the target's activity of interest.

Combinatorial chemistry was first conceived in 1984. Initially, thefield focused primarily on the synthesis of peptide and oligonucleotidelibraries. In 1984 H. Mario Geysen and his group developed, a techniquefor synthesizing peptides on pin-shaped solid supports. In 1985, RichardA. Houghten, developed a technique in which tiny mesh packets, act asreaction chambers and filtration devices for solid-phase parallelpeptide synthesis.

The field's original predominant focus on peptide and oligonucleotidelibraries began to change about 1991 with the development ofcombinatorial techniques for producing small organic molecules withmolecular weights of about 1,000; a class of compounds in which drugsand other valuable bioactive small molecules are most often found.

Two basic methods are used in combinatorial chemistry solid-phase andsolution-phase methods. Using these methods combinatorial compounds arecreated either by solution-phase synthesis or by producing compoundsbound covalently to solid-phase particles.

Solid Phase Methods

Solid Phase Methods: (Fruchtel J S., and Jung G., 1996, Angew. Chem.Int. Ed. Engl. 35, 17-42; which is incorporated by reference in itsentirety).

Solid-phase synthesis makes it easier to conduct multistep reactions andto drive reactions to completion, because excess reagents can be addedand then easily washed away after each reaction step. Another key factorin favor of solid-phase synthesis is that it makes it possible to usesplit synthesis, a technique developed in 1982. Split synthesis produceslarge support-bound libraries in which each solid-phase particle holds asingle compound, or soluble libraries produced by cleavage of compoundsfrom the solid support. For example in a split synthesis method if youhave 3 compound addition steps with 10 compounds used at each step i.e.10 containers for those compounds. This will generate 10³ compounds.Also if you consider all the reaction steps included in a synthesis10,000 compounds made via a solid phase methods using a three-stepchemistry may only require about 22 containers for the chemistry andabout 66 liquid handling steps relative to the 10,000 containers and30,000 liquid handling steps. When you combine these advantages of solidphase synthesis with split synthesis a significant level of synergy isachieved.

A potential disadvantage of solid-phase synthesis is that a hydroxyl,amine, carboxyl, or other polar group are typically present on amolecule to be able to attach it to a solid support. This is apotentially undesirable constraint on the structure of compoundssynthesized on solid phase, because products retain the polar group evenafter they are cleaved from the support. Several groups, have devisedtraceless linkers that avoid this problem, because the linkers areremoved completely from products during the cleavage process. Forexample, an acylsulfonamide linker that can be displaced by variousnucleophiles to add diversity to a library has been described. Anotheralternative to the traceless linker has been developed using achemistry, in which reagents used to cleave products from the solidsupport are incorporated into the product. Using this method a singlecompound on a solid phase can give rise to a chemical series based onthe reagents used to release the products. This can be achieved via theuse of substoichiometric amounts of different cleaving reagents,sequentially reacted with a compound that is synthesized on areact-and-release type resin, and each product is individually eluted.This protocol has advantages when combined with automated chemistrysystems such as those used for peptide and oligonucleotides synthesis.The number of compounds generated with this method can be up to 10 timesthe number of chemistries generated on the solid phase. The result isalso a relatively pure product in solution. This method is an example ofthe combinations of solid and solution phase chemistries.

In order to solve one of the problems caused by the use of splitsynthesis methods, namely knowing which compound in the library showsactivity, encoded libraries have been constructed. An example of anencoding technique is one based on inert halogenated compounds that areused to record the chemical reaction history of each support bead. Thetags can be analyzed by capillary gas chromatography with electroncapture detectors and autosamplers to rapidly reveal the identity ofactive compounds in the library. In addition to this method compoundscan be released from the bead and analyzed by MS and/or GC/MS. Otheralternatives to the deconvolution of the library are based on theresynthesis of sub set pools from a positive hit of pooled compounds.One exciting approach to this problem of compound identification (from ascreen of pooled compounds), is based on the use of affinity selectionplus size exclusion chromatography (to separate bound compounds fromthose that have little or no affinity for the target protein), followedby mass spectroscopy, to identify leads that bind to the target ofinterest. This method eliminates the need to encode the library andmakes use of the molecular weight of the compound as the tag. Someproblems may be encountered from redundancy of some molecular weightswithin a library, but higher resolution and fragmentation MS methods canbe used effectively. In addition combinations of these approaches can beconsidered where the tag is left attached to the compounds which bind tothe target molecule of interest and are then selectively eluted andsubjected to cleavage releasing the tag or code which can then beidentified by MS or GC/MS methods (Karet G, Drug Discovery andDevelopment, Jan 1999, 32-38, www.rdmad.com/drug; which is here byincorporated by reference in its entirety)

Solution Phase Methods

Solution phase chemistry is favored by many for library construction dueto the wider range of organic reactions available for solution-phasesynthesis, the technology used traditionally by most synthetic organicchemists, and products in solution can be more easily identified instandard drug target assays and characterized. A problem forsolution-phase synthesis of one molecule at a time is the finalpurification that can be both expensive and slow. Chromatography iscommonly a first resort since it usually works. In addition, theproblems associated with solution chemistry are compounded whenattempting to make tens of thousands of compounds to generate a libraryor a ‘book’ for a library.

In the generation of libraries of chemistries numerous methods have beendevised resulting in the wide spread use of large libraries of chemicalsto readily allow the discovery of potential drug candidates. Thegeneration of chemical libraries that are free in solution is typicallythe goal of most of the pharmaceutical industry. This aim is due to thenature of many of the drug targets and the associated assays. Also theconstruction and utility of chemical libraries is typically facilitatedbut the generation of master plates of compounds in solution to form thebasis of the chemical library. Thus the general advantages of the solidphase synthesis methods are typically not fully realized in the contextof the current drug discovery efforts. The main reason for this is theinterest not in binding of the compound to the drug target but todemonstrate that the activity of the drug target is altered, whichtypically requires compound free in solution. Further concerns withlibraries of compounds on a solid phase arise from concerns of thepotential influence of the linker and steric effects on the compoundsbound to the solid phase.

Thus methods for the discovery of compounds which bind to targetmolecules is known in the art. Also, the optimization of the initiallydiscovered compound is well known in the art where the affinity isimproved by generation of a pool of related compound via a moreselective combinatorial chemistry approach.

The present invention provides a mechanism to overcome these problems indrug and small molecule discovery.

Embodiments of the Subject Invention Compounds Active on 5-lipoxygenaseas Anti-asthmatics

Screening for Target Protein Binding Elements

Initially a target protein is selected, for example 5-lipoxygenase whichis a molecule involved in inflammatory reactions especially in asthma.Target protein for the subject invention come from numerous fields wheresmall molecules are used to achieve modulation of a biological system ineukaryotic organisms. Examples of such fields are insecticides,fungicides, antivirals, herbicides, anti-parasitics and herbicides whenapplied to humans, animals and plants.

The target protein is then either purified from a natural source inorder to provide sufficient material for the screen or expressed viarecombinant methods to provide sufficient material for the screens.

The target protein is then either labeled directly with a detectablespecies such as a radioactive, electrochemiluminescent, andchemiluminescent or fluorescent label or with an indirectly detectablespecies such as an enzyme, or particle. Alternatively an antibody orequivalent with binding activity to the 5-lipoxygenase is labeled.

The next step is to buy a library of compounds for screening. A libraryof from 1,000 to 1,000,000 is typical of the size that is screened.These are available from a series of companies as described earlier.These libraries of compounds are used to screen for the binding of thetarget protein 5-lipoxygenase. Ideally compounds are bought still boundto the solid phase or are screened for binding directly to immobilizedtarget protein 5-lipoxygenase using methods as described below forscreening.

It is also possible to generate a chemical library of various potentialbinding molecules bound to a solid phase following conventional methodsto give rise to differing potential compounds. The optimal methods forthe construction of the chemical library is to employ the methods ofsplit synthesis coupled to the solid phase (as outlined above). Thelibrary is generated using a series of solid phase chemistries such asto give rise to various ‘chemical books’ that in compilation form thebasis of a library. The library is screened in the form of a library orin the form of the ‘chemical books’. Typically one would take theproducts from the split synthesis and pool the solid phase and use thisas the basis for the screen.

To the pool of beads used as the solid phase for the synthesis, amixture of buffer, detergents, salts and blocking agents such as serumalbumin or other proteins are added. This buffer addition step is usedto ‘block’ the beads or solid phase in such a way that any significantnon specific binding of the selected target (5-lipoxygenase) does notoccur. Following this blocking step the beads are washed and followed bythe addition of the 5-lipoxygenase either labeled or not. The beads orsolid phase are then incubated to allow the binding of the targetprotein binding elements to the target, in this case 5-lipoxygenase.Following the incubation of the target molecule to the beads or solidphase the beads are washed and then the binding of the labeled5-lipoxygenase detected directly. In an alternative format, if the5-lipoxygenase is labeled with an indirectly detectable label such as anenzyme, the beads are then placed in to a substrate reaction solution todetect the presence of the enzyme label. In the case of an enzyme label,substrates for these detection methods are based on insolublechromogenic products. In the case where the 5-lipoxygenase is notlabeled and an antibody or equivalent is available, the beads aresubjected to another binding reaction where the antibody or equivalent,is labeled either directly or indirectly as suggested for the labelingof 5-lipoxygenase. It is also possible at this step to not use a labeledantibody or equivalent and to add a further step where the labeledantibody or equivalent is used. These additional steps can be detectedusing the same standard methods known in the art as suggested for thedirectly labeled 5-lipoxygenase.

Following these steps a series of beads are identified and these beadsare selected from the bead population and subject to analysis todetermine the structure of the binding molecule that is able to bind the5-lipoxygenase as in this example. This is achieved by the use of GC/MSor via molecular tags used during the construction of the library asdescribed earlier. Alternatively a pool which was positive is re-madegenerating a series of sub pools for screening and further re-synthesisand dividing out of the various pooled compounds until a single compoundis presented in a single well for analysis allowing the determination ofthe active compound.

Addition of the Ubiquitination Recognition Element

At this point in the compound discovery path for the subject invention,the target protein-binding element of the compounds of the invention hasbeen identified. These optimal binding molecules are then subjected tofurther chemistry to add the ubiquitination recognition element.

An alternative approach to the discovery of the target protein-bindingelement is based on solution phase screening. In such an examplecompounds (available either via synthesis, natural products or fromcompanies such as ArQule (www.arqule.com), Pharmacopeia(www.pharmacopiea), and Cerep (www.cerep.com) are obtained and added tothe target protein of interest and then subjected to size exclusion toremove the unbound compounds. The protein bound fraction is thensubjected to GC/MS to identify the molecules. In this way the solutionphase screening is made rapid and facile for compounds in solution.

Compounds Active on IL-4 Receptor as Anti-Asthmatics

Introduction

A further embodiment of the subject invention is the development of acompound targeted to a receptor involved in development of asthma. Inrecent studies into the pathophysiology of asthma, IL-13 has beendemonstrated to be the central mediator acting through the IL-4receptor. Thus asthma can be controlled by the lowering of either theIL-13 or IL-4 receptor. The IL-4 receptor consists of two subunits; a140 kd alpha subunit, which binds IL-4 or IL-13 and transduces theirgrowth-promoting and transcription activating functions and a gamma csubunit, common to several cytokine receptors, which amplifies signalingof IL-4 receptor alpha. In this application of the subject invention thetarget for drug development is the IL-4 receptor alpha chain. The IL-4receptor alpha chain has a large intra cellular protein domain thatforms the specific molecular target of the discovery approach of thesubject invention.

Expression of the IL-4 Receptor Alpha Chain

Initially the IL4 receptor alpha (IL-4a) chain intra-cellular domain iscloned from human blood lymphocytes. The cloned DNA is engineered togenerate a gene sequence that directs the expression of the cytoplasmicdomain of the IL-4a chain. This gene sequence is also engineered toinclude a sequence tag that allows the purification and detection of theexpressed receptor sub-unit. This expression is carried out usingvarious methods known in the art. Methods for expression of proteins,are numerous; an example of one is one of the vectors from Invitrogen(Carlsbad, Calif.) such as the His-Patch ThioFusion, which allows forthe optimal expression of proteins in a soluble form and containing aHis tag which allows rapid purification. This system allows for theproduction of soluble protein after cleavage using the enterokinasecleavage site in the cloning vector pThioHis A, B, C. An alternativeXpress system also provides a useful expression system which allowsrapid purification via a His sequence and also a protease cleavage siteto yield the protein of interest with out the His sequences. One of thevectors from the Xpress system, pTrcHis2 A, B, C series is especiallyuseful; this vector allows the use of the His sequence for purificationbut also allows for the tagging of the protein with a myc epitope fordetection and assays for the expressed protein containing the epitopetag sequence myc with an anti-myc antibody. Expression vectors are alsosupplied by other vendors such as New England Biolabs (Beverly, Mass.)whose pMAL-c2 and pMAL-p2 vectors provide an expression system for E.coli which provides a tag which is maltose binding protein (MBP), thistag can be used in purification and also in detection of the fusionprotein. The MBP can be removed by the use of the factor Xa cleavagesite.

Following the cloning of the IL-4a cytoplasmic domain, using art knownmethods for the cloning and expression of proteins, the recombinantprotein is expressed and purified using the tag sequence attached duringthe cloning. This purified receptor sub-unit is then subjected toscreening against a chemical library.

Screening for Binding Molecules from Chemical Libraries

The step of screening for specific molecules is made easy in thisinvention as only binding activity is desired and not specificmodulation of the target protein as is required in traditional drugdiscovery.

The next step is to buy a library of compounds for screening. A libraryof from 1,000 to 1,000,000 is typical of the size that might bescreened. These are available from a series of companies as describedearlier. These libraries of compounds are used to screen for the bindingof the target protein 5-lipoxygenase. Ideally compounds are bought stillbound to the solid phase or are screened for binding directly toimmobilized target protein 5-lipoxygenasen using methods as describedbelow for screening.

It is also possible to generate a library of from 1,000 to 100,000compounds contained on a solid phase using split synthesis methods asdescribed earlier. This library is constructed using a series ofchemical methods resulting in pools of the solid phase used duringsynthesis which form the basis of the ‘books’ which go to make up thelibrary. In addition at the final chemical coupling step used toconstruct the various books the solid phase pools are stored insub-pools forming ‘chapters’ of the ‘books’ in the libraries. These socalled ‘chapters’ form the basis for screening as they contain not onlypools of compounds but also a known chemical-coupling step used in thesynthesis of the ‘chapters’ of the library.

The library can then be screened using two approaches. In both cases thesolid phase from the chemical library to be screened is subjectedincubation with assay buffers with blocking agents such as for example;proteins (i.e. BSA, gelatin), polyvinylpyrrolidone, ficoll, heparin,detergents (i.e. SDS, Tween, NP40, Triton X-100). This incubation stepis to block the non-specific binding sites on the solid phase used inthe generation of the library and allow the determination of specificbinding events. This initial incubation is an art recognized step invarious binding assays such as ELISA, southerns, westerns etc. Followingthis incubation with blocking agents the protein of interest is thenadded to a buffer which typically has the same composition as thatduring the blocking step but can also be modified using lower or noadditional blocking agents with the exception of the detergents whichare typically always present during a binding reaction.

In one of the screening methods the ‘chapters’ of the various ‘books’following the blocking step are then subjected to binding with thepurified receptor sub-unit. The solid phase from this incubation is thenwashed and subjected to a second binding step with a labeled reagentwhich binds to the tag sequence added to the receptor sub-unit duringthe recombinant engineering for the expression of the receptor sub-unit.Typically an antibody to this tag recognizes the tag sequence; examplesthat are in common use are the myc, flag, and his epitopes. Followingthe incubation with the tag specific binding species the presence of thelabeled binding species is detected by the presence of the label that istypically an enzyme such as alkaline phosphatase or peroxidase. Thedetection step typically makes use of an insoluble chromogenic substratethat is readily detected by eye or by image analysis systems.

In an alternative method soluble substrates can also be used andscreened using ELISA plate readers, eye or other spectrophotometricmethods. In its simplest form the various ‘chapters’ of the ‘book’ fromthe library are screened by eye to look for beads that have developed acolor due to the enzymatic action on the chromogenic substrate. Thesecolored beads indicate that the receptor sub-unit is binding to one ofthe compounds within the ‘chapter’ the next step is to determine ifthese so called positive ‘chapters’ contain specific binding or ifbinding is just to the tag binding reagent or some non-specificactivation of the chromogenic substrate. To achieve this, the positive‘chapters’ are screened with out the specific binding step to thereceptor sub-unit. If these positive ‘chapters’ now become negative orshow significantly reduced signals interms of positive solid phases within the mixture then these are considered to be real positive hits in thescreen. These real positive ‘chapters’ are then subjected tore-synthesis. In this re-synthesis the initial chemical steps to createthe specific binding molecule is unknown only the last chemical couplingstep in the compound synthesis is know, as this formed the last chemicalstep which constructed the ‘chapter’. During the re-synthesis of thepositive chapter the chemical step prior to the last chemical couplingis carried out as in the initial synthesis but the solid phase is notpooled and split for the final chemical coupling but are maintained asseparate pools then subjected to the chemical coupling step know forthat chapter. This re-synthesis results in the formation of a new seriesof solid phase compound pools which have the last two chemical couplingsteps known. This new series of solid phase compound pools are screenedas in the initial screen and positive pools are checked as previouslyfor the binding specificity to identify positive pools. The positivepool(s) now allow the re-synthesis of the pool(s) with the last twosteps for the generation of the compound which specifically binds to thereceptor sub-unit. The positive pools are then subjected to the samecycle of re-synthesis and screening as just described but with the lasttwo chemical coupling steps know the pools are maintained individuallyprior to the last know step. In this way the synthesis of the specificcompound able to bind to the receptor sub-unit is deconvoluted from thechemical ‘library’ and identified.

In an alternative method the positive solid phase is removed from thescreen and collected. These are then subjected to the cleavage reactionwhich removed the specific chemistry from the solid phase followed bythe analysis of the various chemical species using GC to separate theindividual compounds followed by MS to determine the molecular weight.This information coupled with the synthesis methods used is used todetermine the compound identity. After the determination of thesevarious candidate specific binding molecules they are thenre-synthesized and subjected to the binding assay to check if these arethe specific compounds that resulted in the positive solid phases.

Addition of the Ubiquitination Recognition Element

This screening effort following methods and protocols known in the artallows the identification of compounds that bind to the receptorsub-unit. These compounds then form the basis for the development ofcompounds of the invention. These compounds are then subjected tofurther chemistry based on the use of the linker group used in thedevelopment of the solid phase chemistry. To this linker group thevarious ubiquitination recognition chemistries are added. This finalstep of chemistry generates the compound of the invention. The compoundof the invention are then subject to analysis to determine which of thecompounds from the chemical library screen with which of theubiquitination recognition elements is able to function most effectivelyin the targeted ubiquitination and/or degradation. In the case where theubiquitination recognition chemistry is based on the N-end rule therabbit reticulocyte lysate forms the basis for the assay using therecombinant produced receptor sub-unit labeled with for example ¹²⁵I tofollow the fate of the protein. In addition the compounds of theinvention can be tested in a mammalian tissue culture system where thetarget protein either intact or as an engineered fragment is expressed.In such a mammalian tissue culture system the compounds effect on thetarget protein's level is determined by making use of the tag sequencewhich can be engineered into the recombinant expression of the targetprotein during the construction of the mammalian tissue culture testsystem. The tag sequence is used to determine the levels of the targetprotein during the incubation with the potential compounds screened andsynthesized as described above. This assay for the tag sequence can takethe form of a western blot or via an ELISA, for example. Other tagswhich are valuable to use are those based on the green fluorescentprotein, which allows the analysis of protein levels in living cellsand/or organisms.

The compounds that show the optimal activity in the test systems willthen form the basis for the next stage of drug development. In this nextstage these selected compounds are subjected to the recognized drugdevelopment path. The drug development path determines the potentialvalue of the compounds by evaluating a series of factors includingbioavailability; toxicology, pharmacology and efficacy in animal modelsbefore the compounds are considered for human testing.

Development of Pesticides

An alternative embodiment of the subject invention is the development ofpesticides. Pesticide is a general classification that includesinsecticides, rodenticides, fungicides, herbicides, and fumigants. Theaim of the pesticide is the destruction of some life form and as suchselectivity is desirable. The methods that have been described for thesubject invention for development of active compounds to the5-lipoxygenase and IL-4 Ra also apply to the development of pesticides.Pesticides are also compounds of the invention, which are targeted to animportant biochemical pathway in a pest that is required for itssurvival or prolonged viability. A pest is an organism that has somedirect or indirect deleterious effect on mankind. The term pest iswidely used to cover any organism that has some direct or indirectdeleterious effect on mankind. Some examples of pests are aphids, moths,lice, fleas, locusts, mice, rats, weeds etc. In the development of apesticide the methods outlined above are followed with the exception ofthe target in the case of developing pesticides key biochemical pathwaysfor survival in the pest would form the basis of the molecular targetselected to screen for protein binding elements. In the case ofinsecticides examples of key biochemical pathways include, ecdysone20-monooxygenase, ion channel of the GABA gated chloride channel,acetylcholinesterase, voltage-sensitive sodium channel protein, calciumrelease channel, and chloride channels.

The subject invention is ideally suited to the optimal development ofpesticides due to subject inventions ability to rapidly screen forspecific interactions which can be developed into a highly speciesspecific compounds of the invention. Specificity is of prime importancefor the development of pesticides as the targeted pests are eitherpresent in close proximity to mankind, or as in the case of agriculturepests, the pests are targeted on food intended for mankind where toxiccompound residues are unacceptable. For example, in the case ofpesticides that are targeted to kill aphids, the compound is ideallytargeted only to the aphids and has no effect on the beneficial insectssuch as ladybugs, and bees. The subject invention provides through itsmolecular basis of compound selection, both a facile and an improvedmethod for the development of pesticides. In the development of anoptimal pesticide the specific target protein involved in the keybiochemical pathway is cloned and engineered using well known methods togenerate a source of protein for screening the chemical compound librarywhich has sequence tags to enhance the screening and characterization ofthe compounds of the invention. This process is also repeated using theproteins from the organisms also posses the same critical biochemicalpathways but are not the target pests. Thus a set of proteins can bedeveloped from the pest organism and from organisms which are likely tobe exposed to the compounds of the invention when used as pesticides. Inthe screening procedures as described earlier for the development of theanti-asthmatic compounds in addition to the screen for binding to thedesired target, absence of binding can also be screened for using theproteins from the non-pest organisms. In this way a set of compounds canbe selected which show specificity to the target pest organism and notcommonly encountered or related non-pest organisms. This type ofscreening is an advantage of the subject invention as it is based on theuse binding and does not require a complex activity assay. In this waythe subject invention provides for a low cost and rapid route to theselection of molecular species which have a high degree of speciesspecificity.

The subject invention allows for the development of selectivepesticides. The development of pesticides follows many of the previouslydescribed methods. The screen methods for the binding molecules thatrecognize the specific target have been described earlier for the IL-4receptor alpha chain and 5-lipoxygenase. These screens are used in orderto find molecules with the binding affinity for the target proteins ofinterest; for example to develop a specific herbicide the proteinenolpyruvylshikimate-phosphate synthase represents a good target as thisis the molecular target for glyphosate. The target protein is eitherpurified from the natural source or cloned and expressed using variousrecombinant methods to produce the enolpyruvylshikimate-phosphatesynthase. An ideal target for the development of a selective herbicideis poison ivy, in this case the enolpyruvylshikimate-phosphate synthasefrom poison ivy is used as the source of the target protein. The screenfor the binding molecules is initially focused on this target butsecondary screens are carried out on the various other plantsenolpyruvylshikimate-phosphate synthase normally present in the sameenvironment as poison ivy is found growing naturally. The secondaryscreen is used to establish the binding molecules that do not bind tothe other plant enolpyruvylshikimate-phosphate synthases in order toprovide the level of specificity desired. Following the identificationof the selective binding molecule this is then coupled to theubiquitination recognition element in order to generate the herbicide ofthe subject invention, which is selective to poison ivy. The developmentof the selectivity is further enhanced using comparative sequencing ofthe various molecular targets thus defining the various sequenceelements that are unique to poison ivy (or other target organism). Thesequence information then allows both the definition of the molecularbinding site within the molecule but also the sequences of the variousproteins that are used in the secondary screens to define thespecificity of the final binding molecules from the screen. It will beunderstood that the methods described in the subject invention benefitgreatly from the recent advances in genomic sequencing which make muchof the sequence information readily available or easily obtainable. Itwill be understood by those skilled in the art that these methods can beapplied readily to any pest in the development of pesticides. In thecase where no molecular target is known or can be defined it will beunderstood that the subject invention also allows a route to discoveryof such molecular targets. This discovery can be achieved through theselection of targets by various levels of homology with know targets orvia selection based on no homology which leads more rapidly to thedevelopment of selectivity even if it takes longer to define the roleand value of these new molecular targets. Other examples of targets forthe development of herbicides include, Acetyl-CoA carboxylase,adenylosuccinate synthetase, protoporphyrinogen oxidase, andenolpyruvylshikimate-phosphate synthase.

Development of Compounds Effective Against Parasites

In the development of compounds that are targeted to parasitic organismsthe current invention provides for significant advantages. It has beentraditionally a problem developing drugs that provide for the selectivetoxicity to various parasites of mankind and his domestic animals. Thisproblem has been largely due to the problems of culturing theseorganisms and to the problems of finding a toxin that has the desiredlevel of toxicity to the parasite with out damaging the host organism.This problem has presented itself due to the large number of relatedbiochemical pathways that are shared between the eukaryotic organisms.Some efforts have been made with some success to define biochemicaldifferences this has not yielded a broad range of targets for thedevelopment of drugs. The subject invention provides for a more facileand optimal method for the development of compounds effective againstparasites in the groups of Protozoan parasites: Balantidium,Cryptosporidium spp., Giardia spp, Plasmodia, Trypanosoma, Leishmania,Trichomonas, Entamoeba, Eimeria, Toxoplasma, Plasmodium, Babesia,Theileria, Metazoan parasites: Nematode parasites, Ascaris spp.,Capillaria spp., Dracunclus spp., Enterobius spp., Filariasis due tovarious organisms, hookworm infections, Strongyloides spp., Toxocaraspp., Trichinella spp., Trichuris spp., Taenia spp., Diphyllobothriumspp., Hymenolepis spp., Echinococcus spp., Shistosoma spp., Fasciolopsisspp., Heterophyes spp., Metagonimus spp., Clonorchis spp., Opisthorchisspp., Paragonimus spp. etc.

Targets for compound development: Leishmania, proteins of the sterolsynthesis pathway: Plasmodium, dihydrofolate reductase; dihydrofolatereductase-thymidylate synthase (bifunctional) resistance known due tomutations in the gene for this enzyme, heme polymerase: Trypanosoma,ornithine decarboxylase, trypanothione reductase, Ornithinedecarboxylase of the trypanosoma represents an desirable candidate fordestruction due to its long half-life and low turn over in trypanosoma.

Protein Level Control

This invention is also to a method for the control of protein levelswith a cell. This is based on the use of compounds of the inventionwhich are known to interact with a specific protein or protein sequenceelement. These specific proteins known to interact with compounds of theinvention are used to generate chimeric fusion proteins with a desiredtarget protein. These chimeric fusion proteins thus functionally linkthe ability to be destabilized by the compounds of the inventions to thedesired target protein. In this way known compounds of the invention andknown proteins and/or protein sequence elements can be combined totarget the genetic engineering of another protein to render itdegradable and thus controllable by a compound of the invention. Thefollowing are by way of illustration of some possible application ofthis idea.

Control of Protein Levels Within a Cell

In another embodiment of the subject invention, control of specific geneproducts is achieved. In this embodiment a gene(s) is engineered suchthat its expression results in the production of the desired protein butwith the addition of a protein which has a specific binding affinity fora small molecule. Examples of such sequences are streptavidin, avidin,antibodies, single chain antibodies, thioredoxin, maltose bindingprotein, and the peptide motif CCXXCC (SEQ ID NO 47), andWEAAAREACCRECCARA (SEQ ID NO 48), (Griffin B A, 1998, Science 218, 269).In the case of thioredoxin and the peptide motif CCXXCC (SEQ ID NO 47),WEAAAREACCRECCARA (SEQ ID NO 48), and AEAAAREACCRECCARA (SEQ ID NO 49),these are known to bind to tightly to organoarsenical compounds. Onepotential binding species for the peptide motif CCXXCC (SEQ ID NO:47)and WEAAAREACCRECCARA (SEQ ID NO 48), and AEAAAREACCRECCARA (SEQ ID NO49), is 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein with otherbis-organoarsenical being useful (Griffin B A, 1998, Science 218, 269,which is hereby incorporated by reference in its entirety).

Having generated the modified gene for the protein of interest thesegenes are then introduced into the cells desired either forming thebasis of a cell culture study in vitro or through the generation of atransgenic animal which expressed the modified gene in its normalcontext or aberrantly to determine its role within the intact organism.An example of this type of engineering is described in Griffin B A,1998, Science 218, 269.

In this embodiment the compound of the invention is built around thesmall molecule with a specific binding affinity for a specific aminoacid as exampled above. In the above example these are biotin bindingwith streptavidin and avidin, any small molecules binding with singlechain antibodies such as biotin, digoxin, fluorescein and theorganoarsenical compounds such as4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein, p-aminophenylarsineoxide binding with thioredoxin and the peptide motif CCXXCC (SEQ ID NO47), WEAAAREACCRECCARA (SEQ ID NO 48), and AEAAAREACCRECCARA(SEQ ID NO49). To these binding molecules are attached the ubiquitinationrecognition elements to generate a bifunctional molecule which is ableto bind to the genetically engineered protein and activate theubiquitination of the engineered protein. Having generated thesebifunctional molecules these then are used to treat the cells and/ororganisms which contain the engineered protein. This treatment resultsin the rapid degradation of the engineered protein in a dose dependentfashion allowing the determination of the role of the various proteinsin the biology and/or physiology of the cell and/or organism. Thisembodiment of the subject invention allows the rapid generation of aseries of mutant proteins, making use of an identical compound andtreatment schedule in affecting changes within a cell and/or an organismthat allows for optimal determination of the role of various proteins inan controlled study. This is achieved with less perturbation of the celland/or organisms natural biochemistry than is possible with othermethods.

Control of Green Fluorescent Protein Levels

An example of the above embodiment is directed to the demonstration oftargeted ubiquitination to mediate degradation of a protein insideliving cells. The green fluorescent protein (GFP) ECFP plasmid vector(Clontech, Palo Alto, Calif.) was chosen in order to engineer thefollowing binding site AEAAAREACCRECCARA (SEQ ID NO 49), into the Cterminus of the expressed ECFP (GFP) following established methods toform an expression vector able to direct the expression a GFP with a Cterminal tagged end ECFP-Cys4 (Griffin B A, et al 1998 Science, 281,269-272). This choice of the ECFP was also made so that the formation ofthe complex of 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein with theECFP-Cys4 demonstrates fluorescent energy transfer (FRET) from ECFP-Cys4to the bound 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein. This vectorwith the ECFP-Cys4 gene is then transfected into HeLa cells anddemonstrates that expression is obtained and the protein had theexpected long half life of >20 hrs. Various compounds of the inventionare made as follows; with 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluoresceincoupled using EDC chemistry, to a ubiquitin recognition elementsselected from Arg-εAhx-Lys; Arg-β-Ala-εAhx-Lys; Arg-εAhx-εAhx-Lys;Phe-εAhx-Lys; Phe-β-Ala-εAhx-Lys; Phe-εAhx-εAhx-Lys, orp-aminophenylarsine oxide coupled using EDC chemistry, to a ubiquitinrecognition elements selected from Arg-εAhx-Ala; Arg-β-Ala-εAhx-Ala;Arg-εAhx-εAhx-Ala; Phe-εAhx-Ala; Phe-β-Ala-εAhx-Ala; Phe-εAhx-εAhx-Ala.These compounds of the invention molecules are then added to the cellstransfected with the ECFP-Cys4 expression vector and subsequentlytreated with 100 ug/ml cyclohexamide to block further protein synthesis.The fluorescence is measured over time to determine the levels of theprotein and the protein bound to the compounds of the invention, usingexcitation at 440 nm and emission at 480 nm to look at ECFP levels and635 nm when the 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein basedcompounds are used. The stimulation of degradation seen with thep-aminophenylarsine oxide based compounds was observed by drop influorescence of the ECFP relative to control cells. In the case of thestudies with the 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein basedcompounds an initial rise of the FRET signal at 635 nm is seen followedby a drop in the signal compared to controls where only4′,5′bis(1,3,2-dithioarsolan-2-yl)fluorescein is used to treat thecells. In addition to these fluorescence studies, the levels of proteinusing western blot analysis is examined using an antibody to the ECFP,GFP (Clontech, Palo Alto, Calif.), which demonstrated that compounds ofthe invention lower the levels of the ECFP. Concentrations for thevarious compounds and other molecules used were from 0.1 uM to 100 uM.This study showed the ability to use the targeted ubiquitination toalter the levels and half-life of a protein in a living cell usingcompounds of the invention.

Control of Protein Levels in the Liver of a Transgenic Organism

An example of the above embodiment is the demonstration of targetedubiquitination to mediate quantitative and tissue specific control ofgene expression in transgenic mice. The expression vector wasconstructed using the luciferase gene and a liver specific promoter˜thepromoter of the liver enriched activator protein driving the expressionof the luciferase gene (Kistner A., 1996, Proc. Natl. Acad. Sci. 93,10933-10938). The luciferase gene was engineered to contain theAEAAAREACCRECCARA (SEQ ID NO 49), sequence at the C terminus usingsynthetic oligonucleotides and PCR based cloning. The final expressionvector consisted of the P_(LAP), promoter driving the expression of theluciferase gene containing the AEAAAREACCRECCARA (SEQ ID NO 49),sequence (the binding site for the compounds of the invention). Thisexpression vector was then used to generate transgenic mice. Transgenicmice lines were generated by pronuclear injection using standardtechniques and analyzed by Southern blot using a BamHI-EcoRV fragment ofthe luciferase gene (Kistner A., 1996, Proc. Natl. Acad. Sci. 93,10933-10938). The tissue specific expression of the modified luciferasegene was demonstrated using standard methods on liver, pancreas, kidney,stomach, muscle, thymus, heart, and tongue. In order to modulate thelevels of the luciferase gene the transgenic mice were injected with4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein coupled using EDCchemistry, to a ubiquitin recognition elements selected fromArg-εAhx-Lys; Arg-βAla-εAhx-Lys; Arg-εAhx-εAhx-Lys; Phe-εAhx-Lys;Phe-β-Ala-εAhx-Lys; Phe-εAhx-εAhx-Lys, or p-aminophenylarsine oxidecoupled using EDC chemistry, to a ubiquitin recognition elementsselected from Arg-εAhx-Ala; Arg-β-Ala-εAhx-Ala; Arg-εAhx-εAhx-Ala;Phe-εAhx-Ala; Phe-β-Ala-εAhx-Ala; Phe-εAhx-εAhx-Ala, which formed a setof compounds of the invention. The serum concentrations achieved arefrom 1 micromolar to 1 millimolar. The levels of luciferase activity arelowered as the doses of the various compounds are increased. Thisresponse was also seen when the study was carried out using liver slicesin vitro using similar concentrations in the tissue culture medium usedfor the liver slice incubations.

Control of the Physiology a Transgenic Organism

An example of the above embodiment is the analysis of the effect ofexpressing CaMKII on specific forms of memory. CaMKII is aserine-threonine protein kinase expressed primarily in neurons of theforebrain. The ability of CaMKII to become persistently active inresponse to a transient Ca stimulus indicates its potential involvementin memory. Mutation of the Thr286 to Asp in CaMKII (CaMKII-Asp286)produces a calcium-independent form that mimics the auto-phosphorylatedform. The transgenic expression of CaMKII-Asp286 leads to a shift inresponse to stimulation as well as a severe defect in spatial memory. Toobtain tissue-specific and ubiquitin regulated degradation a line ofmice is generated expressing the CaMKII-Asp286 tagged with aAEAAAREACCRECCARA (SEQ ID NO 49), sequence (CaMKII-Asp286-tag) undercontrol of the native CaMKII promoter to ensure natural tissue specificexpression. In addition a line of mice was also constructed expressingbeta-galactosidase tagged with a AEAAAREACCRECCARA (SEQ ID NO 49),sequence (beta-gal-tag) under control of the native CaMKII promoter.These mice both demonstrated forebrain-specific expression. Severedefects in spatial memory were observed in response to CaMKII-Asp286-tagexpression using the Barnes circular maze. The treatment of these micebrains with the 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein,p-aminophenylarsine oxide coupled to a ubiquitin recognition elementsselected from Arg-εAhx-Cys; Arg-β-Ala-εAhx-Cys; Arg-εAhx-εAhx-Cys;Phe-εAhx-Cys; Phe-β-Ala-εAhx-Cys; Phe-εAhx-εAhx-Cys, demonstrated areversal of this profound memory impairment. In the mice with thebeta-gal-tag expression treatment of both the mice and tissues from thefore-brain with 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein,p-aminophenylarsine oxide coupled to a ubiquitin recognition elementsselected from Arg-εAhx-Cys; Arg-β-Ala-εAhx-Cys; Arg-εAhx-Ahx-Cys;Phe-εAhx-Cys; Phe-β-Ala-εAhx-Cys; Phe-εAhx-εAhx-Cys, demonstrateddramatic reductions in the levels of beta-galactosidase activity. Thissystem has the distinct advantage over the currently available systemswhich are based on multiple gene products, unnatural and modifiedpromoter elements, requiring multiple rounds of transfection and thescreening of multiple clonal cell lines to identify the desired cellline from each transfection; for example the Tet-Off™ and the Tet-On™gene expression system (U.S. Pat. No. 5,464,758) sold by Clontech (PaloAlto, Calif.; www.clontech.com).

Control of Gene Expression

In an extension of the above embodiments, relating to the control ofgene expression. The tag sequence or its equivalent can be geneticallyengineered into the coding sequence of various transcription and/ortransactivating factors to render their protein levels within the cellsensitive to the presence of a small molecule activator of theubiquitination pathway. In this way any given transactivating factor (X)can be modified to contain tag sequence as above resulting in theexpression either in the native tissue or other-wise via themodification of said transactivating factors promoter and/or operatorand/or enhancer region, to allow the expression of X-tag. The levels ofthe X-tag protein can then be controlled via the use of a small moleculeactivator of the ubiquitination pathway in order to affect theexpression of any given gene dependent on said X for control and thusits protein product, in order to determine its role or to control someother aspect of the cell or organisms biochemistry, physiology or formthough the modification of gene expression. An example is atransactivating factor that controls multiple proteins expressionlevels. Control of this single transactivating factor results in theeffective control of multiple proteins via a small molecule activator ofthe ubiquitination pathway.

Control of Steroid Production in Genetically Engineered Animals

A ramification of the proceeding embodiment is the possibility ofgenerating modified cells and organisms which contain either a singleprotein or multiple proteins modified with a selective binding domainwhich allows the control of a specific gene with the cells or organismsto give rise to a desired biological effect. For example the reductionof boar taint in pigs can be achieved by the removal of the hormoneGnRH. This embodiment of the subject invention allows for themodification of the GnRH receptor to allow its targeted degradation inthe presence of a compound of the invention. In this way boar taint canbe controlled by feeding a compound of the invention, that downregulates the receptor resulting in the reduction of steroidbiosynthesis responsible for boar taint.

Control of Flower Color in Genetically Engineered Plants

In a further example, a gene for the biosynthesis of a flower color ismodified, allowing expression of a functional protein tagged with thespecific binding sequence. This expression of a modified proteininvolved in the biosynthesis of flower color, such as the genes involvedin the biosynthesis of flavonoids, carotenoids and anthocyanins; i.e.flavanone 3-hydroxylase, anthocyanin synthase, dihydroflavonol4-reductase, flavonoid 3′,5′-hydroxylase, anthocyanin 5-aromaticacyltransferase, UDP-glucose:flavonoid 3-O-glucosyltransferase,anthocyanin rhamnosyltransferase, anthocyanin 3′-methyltransferase,anthocyanin 3′5′-methyltransferase, leucoanthocyanidin dioxygenase,anthocyanidin synthase anthocyanin acyltransferase, chalcone synthase,chalcone flavanone isomerase, glutathione S-transferase, allows for themodification of flower color by addition of the compounds of theinvention specific for the modified biosynthetic protein. In addition tothe proteins involved in the synthesis of flower color, the geneproducts involved in the regulation of the expression of the synthasesand other proteins involved in the production of flower color are alsoconsidered targets of the subject invention. Examples of the regulatorygenes include the R and C1 gene families, an2 and jaf13, the delilagene. Quattrocchio F. 1998, Plant J. 13(4), 475-488.

Resistance Control in Genetically Engineered Plants

In a still further example of the above embodiment of the subjectinvention relating to the selective control of protein levels to achievea desired biological response. The gene involved in the herbicideresistance to glyphosate (Roundup®) in Roundup Ready® soybeans theenolpyruvylshikimate-phosphate synthase from the bacteria agrobacteriumsp. Strain CP4 (CP4EPSPS), is engineered with a gene sequence encoding asmall molecule binding sequence i.e. tag as described above, whichallows the activation of the targeted degradation of the herbicideresistance marker using compounds of the invention. In this waytransgenic plants containing the engineered resistance gene CP4EPSPS canbe rendered sensitive to the herbicide glyphosate by contacting thetransgenic plants with compounds of the invention.

Gene Expression Control in Gene Therapy Vectors

In a further example of the selective control of protein levels it iscontemplated that the genes for pre-selected proteins are engineered tocontain the coding sequence for a small molecules binding sequence. Thusrendering the protein, expressed from the engineered genes of thepre-selected amino acid, targets for compounds of the invention thatallows these proteins activity and/or levels to be controlled by thecompounds of the invention. The engineered genes of the pre-selectedamino acid are then cloned into vectors for gene transfer into a host.

In the case of human gene therapy vectors that are useful are virusessuch as; adenovirus, retroviruses, herpes virus, vaccina virus. In thecase of other organisms potential vectors for gene therapy are selectedfrom the viruses which are known to infect these host or can be modifiedto infect these hosts. In addition to these viral vectors which offersignificant efficiencies, native DNA or RNA (not in the context of aviral genome) are also useful for gene therapy. In the case of DNA thecloned gene for the pre-selected protein containing the smallmolecule-binding site is placed in the DNA sequence such that it isunder control of suitable transcription control elements. Thisengineered DNA is then administered to the organism in such a way thatDNA is taken up by cells efficiently resulting in the DNA being eithertranscribed and translated directly or integrated into the genomefollowed by transcription and translation. Typically DNA and RNA uptakeinto cells is poor and this is typically stimulated by the use ofvarious chemical and physical methods. Examples of chemical methods arethe use of liposomes, calcium phosphate, detergents, ion-exchangecompounds such as DEAE dextran, and also methods linked to specificreceptors such as the folate receptor via linkage to folate analogues.The physical methods that have proved valuable for getting DNA into acell are electroporation, heat, physical membrane perturbation such aspricking, and scrapping of cells.

Pharmaceutical Preparations of the Compounds of the Invention

The pharmacologically active compounds of the subject inventionsoptionally are combined with suitable pharmaceutically acceptablecarriers comprising excipients and auxiliaries which facilitateprocessing of the active compounds. These are administered as tablets,dragees, capsules, and suppositories. The compositions are administered,for example, orally, rectally, vaginally, pulmonary or released throughthe buccal pouch of the mouth, and are optionally applied in solutionform by injection, orally or by topical administration such astransdermal patchs. The compositions may contain from about 0.1 to 99percent, preferably from about 50 to 90 percent, of the activecompound(s), together with the excipient(s).

For delivery of high molecular weight compounds and compounds with poorbioavailability of the subject invention methods based on various knownformulations and methods are contemplated; these include the use ofantibodies, pyridoxyl, insulin, transferrin, galactose, sialyl-LewisX,liposomes, asialolglycoprotein, folate, invasin, iontophoresis,galparan, transportan, homeobox peptides (such as those based onantennapedia residues 43-58), for intracellular delivery.

For parenteral administration by injection or intravenous infusion, theactive compounds are suspended or dissolved in aqueous medium such assterile water or saline solution. Injectable solutions or suspensionsoptionally contain a surfactant agent such as polyoxyethylenesorbitanesters, sorbitan esters, polyoxyethylene ethers, or solubilizing agentslike propylene glycol or ethanol. The solution typically contains 0.01to 5% of the active compounds. The active compounds optionally aredissolved in pharmaceutical grade oils (ie vegetable, synthetic) forintramuscular, sub-cutaneous or sub-dermal injection. Such preparationscontain about 1% to 50% of the active compound(s) in oil. Also theactive compounds optionally are incorporated into or onto particulatepreparations of polymeric compounds such as polylactic acid,polyglycolic acid, hydrogels, etc. or into liposomes, niosomes,microemulsions, micelles, unilamellar or multilamellar vesicles,biodegradable injectable microcapsules or microspheres, or proteinmatrices, erythrocyte ghosts, spheroplasts, skin patches, or other knownmethods of releasing or packaging pharmaceuticals.

Suitable excipients include fillers such as sugars, for example lactose,sucrose, mannitol or sorbitol, cellulose preparations and/or calciumphosphates, for example tricalcium phosphate or calcium hydrogenphosphate, as well as binders such as starch paste, using, for example,maize starch, wheat starch, rice starch or potato starch, gelatin,tragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodiumcarboxymethyl cellulose and/or polyvinyl pyrrolidone.

Auxiliaries include flow-regulating agents and lubricants, for example,silica, talc, stearic acid or salts thereof, such as magnesium stearateor calcium stearate and/or polyethylene glycol. Dragee cores areprovided with suitable coatings which, if desired, are resistant togastric juices. For this purpose, concentrated sugar solutions are used,which optionally contain gum arabic, talc, polyvinyl pyrrolidone,polyethylene glycol and/or titanium dioxide, lacquer solutions andsuitable organic solvents or solvent mixtures. In order to producecoatings resistant to gastric juices, solutions of suitable cellulosepreparations such as acetylcellulose phthalate orhydroxypropylmethylcellulose phthalate are used. Dyestuffs or pigmentsare optionally added to the tablets or dragee coatings, for example, foridentification or in order to characterize different compound doses.

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral useare obtained by combining the active compound(s) with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

Other pharmaceutical preparations which are useful for oral deliveryinclude push-fit capsules made of gelatin, as well as soft-sealedcapsules made of gelatin and a plasticizer such as glycerol or sorbitol.The push-fit capsules contain the active compound(s) in the form ofgranules which optionally are mixed with fillers such as lactose,binders such as starches and/or lubricants such as talc or magnesiumstearate, and, optionally stabilizers. In soft capsules, the activecompounds are preferably dissolved or suspended in suitable liquids suchas fatty oils, liquid paraffin, or polyethylene glycols. In addition,stabilizers optionally are added.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water soluble form, for example,water soluble salts. In addition, suspensions of the active compounds asappropriate in oily injection suspensions are administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortri-glycerides. Aqueous injection suspensions optionally includesubstances which increase the viscosity of the suspension which include,for example, sodium carboxymethylcellulose, sorbitol and/or dextran. Thesuspension optionally contains stabilizers.

In another embodiment, the active compounds are formulated as part of askin lotion for topical administration. Suitable lipophilic solvents orvehicles include fatty oils, for example sesame oil or coconut oil, orsynthetic fatty acid esters, for example ethyl oleate or triglycerides.

In another embodiment, the active compounds are formulated in vehiclessuitable for direct treatment of gastrointestinal mucosa. Examplesinclude mouthwashes, liquids (solutions or suspensions) to be swallowed,or viscous fluids (e.g. solutions of methylcellulose,carboxymethylcellulose, xanthan gum, etc.) which are administered orallyor rectally.

Other pharmaceutical preparations which are used rectally, especiallyfor treatment of the colon and rectum, include, for example,suppositories which consist of a combination of active compounds with asuppository base. Suitable suppository bases are, for example, naturalor synthetic triglycerides, paraffin hydrocarbons, polyethylene glycolsor higher alkanols. In addition, gelatin rectal capsules which consistof a combination of the active compounds with a base are useful. Basematerials include, for example, liquid triglycerides, polyethyleneglycols, or paraffin hydrocarbons.

Other pharmaceutical preparations which are used orally, especially fortreatment of the lungs, trachea, sinus and oral cavity, include, forexample, powders, foamates, nano-particles, liposomes, niosomes,microemulsions, micelles, unilamellar or multilamellar vesicles. Thesemay optionally be administered as for example sprays and aerosols.

The following examples are illustrative, but not limiting of thecompositions and methods of the present invention. Other suitablemodifications and adaptations of a variety of conditions and parametersnormally encountered which are obvious to those skilled in the art arewithin the spirit and scope of this invention.

EXAMPLES Example 1

Targeted Degradation of HIV Integrase

Expression and Purification of His₆-HIV Integrase

Full length HIV-1 IN is expressed in E. coli and purified by theestablished protocols of Craigie, R., Hickman, A. B. and Engelman, A.(1995) in HIV Volume 2: A Practical Approach, pp53-71, J. Karn ed.,Oxford Univ. Press, New York. The pINSD.His plasmid, containing theHIV-_(NL4-3) coding sequence inserted in the pET-15b His₆ expressionvector (Novagen), is available from the NIAID AIDS Research andReference Reagent Program as transformed HB101. pINSD.His is preparedusing a Qiagen plasmid purification kit, and transformed into BL21(DE3)by electroporation for expression in shaker flask cultures by Protocol2, from Craigie, R., Hickman, A. B. and Engelman, A. (1995) in HIVVolume 2: A Practical Approach, pp53-71, J. Karn ed., Oxford Univ.Press, New York. Following Protocol 4, as described in Craigie, R.,Hickman, A. B. and Engelman, A. (1995) in HIV Volume 2: A PracticalApproach, pp53-71, J. Karn ed., Oxford Univ. Press, New York, bacteriaare lysed and the His₆ HIV IN purified under native conditions. Theprotocol is basically a one step chelating column purification of a 2 MNaCl-soluble lysate fraction.

Synthesis of Bifunctional Trans-Targeting Derivatives of L-Chicoric Acid

Compounds are designed based on bromoacetic acid derivatization, viaethylenediamine, of an L-chicoric acid carboxyl group for selectivereaction with thiols. Linkers are composed of aminocaproic acid andβ-alanine, variations of this are readily synthesized by solid phasemethods to include cysteine for conjugation to bromo acetylatedL-chicoric acid. Thiol addition to bromoacetic acid is selective,accomplished under mild reaction conditions, and yields are nearquantitative (Inman, J. K., Highet, P. F., Kolodny, N., and Robey, F. A.(1991) Bioconjugate Chem. 2, 458-463). A significant aspect of thisstrategy is that once L-chicoric acid has been successfullybromoacetylated and purified, any number of different trans-targetingcompounds are obtained easily and in high yield from recognition/linker“cassettes” generated readily from solid phase synthesis.

The synthesis of L-chicoric acid is accomplished following literatureprocedures (Panizzi, L., Scarpati, M. L. and Scarpati, R. (1954) Gazz.Chim. Ital. 84, 806-815, Scarpati, M. L. and Oriente, G. (1958)Tetrahedron 4,43-48., FIG. 2). The bromoacetyl derivatization strategyis to generate bromoacetic acid anhydride for reaction with commerciallyavailable Boc-blocked ethylenediamine (Aldrich) to giveN-bromoacetyl-ethylenediamine after TFA deprotection and crystallization(4, FIG. 3, Scheme 2). 4 is conjugated to L-chicoric acid activated withNHS at one of the symmetrically equivalent carboxylate groups. NHS esteractivation of acid groups for primary amine coupling is used routinelyin conjugation chemistry. Preparative RP HPLC is used for purificationof the desired monoester product from the reaction mixture. Theconjugation reaction to give the final bromoacetyl derivative ofL-chicoric acid, 6, is straightforward in terms of mixture complexityand product purification. NMR and mass spectral analysis monitor allsteps of the synthesis.

The E3α ubiquitination recognition elements are based on the studies byBachmair, A. and Varshavsky, A. (1989) Cell 56, 1019-1032. Theaminocaproic acid (εAhx) and β-Ala will give the ubiquitinationrecognition elements considerably more degrees of freedom than a peptideand are not susceptible to proteinases. Combinations of aminocaproicacid and β-Ala are used to adjust hydrophobic character, flexibility andparticularly the length of the linkers. Changing between Type I (basic)and Type II (hydrophobic) recognition signals significantly affect thehydrophobic character of the trans-targeting compounds, but have aneffect on linker function since these sites are spatially distinct onE3α.

The first series of recognition/linkers include Arg (Type I) and Phe(Type II) recognition components and three different spacer elements;εAhx-Cys, β-Ala-εAhx-Cys, and εAhx-εAhx-Cys with molecular weights ofapproximately 315, 386 and 428, respectively.

Solid Phase Synthesis of E3α Recognition/Linker Components(Ubiquitination Recognition Elements)

Various ubiquitination recognition elements were synthesized by solidphase peptide synthesis and characterized by C₁₈ reverse phase HPLC andMALDI-TOF mass spectral analysis (American Peptide Company, Inc.,Sunnyvale, Calif.). The linker elements include caproic acid (εAhx) andbeta-alanine (β-Ala) for a high degree of freedom of motion, and aC-terminal Cys residue for specific thiol conjugation to targetingmolecule components. The compounds were synthesized to >90% purity in 10mg amounts.

MW Arg-εAhx-Cys 390 Arg-β-Ala-εAhx-Cys 462 Arg-εAhx-εAhx-Cys 521Phe-εAhx-Cys 400 Phe-β-Ala-εAhx-Cys 452 Phe-εAhx-εAhx-Cys 531

Further ubiquitination recognition elements are synthesized as followsusing methods described above.

1. Arg-Ala-εAhx-Cys 2. Arg-Ala-β-Ala-εAhx-Cys (SEQ ID NO:66) 3.Arg-Ala-εAhx-εAhx-Cys 4. Phe-Ala-εAhx-Cys 5. Phe-Ala-β-Ala-εAhx-Cys (SEQID NO:67) 6. Phe-Ala-εAhx-εAhx-Cys

Synthesis of L-Chicoric Acid

FIG. 2, Scheme 1.

To 0.36 g of caffeic acid (Aldrich) in 100 mL of H₂O is added 10 g ofsodium bicarbonate and the solution is cooled to 0° C. A 20% solution ofCOCl₂ in toluene (Fluka) is added slowly with stirring, followed by theslow addition of 20 mL of 6 M HCl. The solid product is filtered undervacuum, washed with H₂O and acetone, and recrystallized from glacialacetic acid to give the blocked catechol of caffeic acid, 3 (Panizzi,L., Scarpati, M. L. and Scarpati, R. (1954) Gazz. Chim. Ital. 84,806-815).

To 0.25 g of 3 in benzene is added 0.30 g of PCl₅ and the reactionmixture is refluxed until 20 min after complete solution, and thenallowed to stand for 1 hr. The solid product is rapidly filtered undervacuum, washed with ether, and dried under vacuum to yield 4 (Panizzi,L., Scarpati, M. L. and Scarpati, R. (1954) Gazz. Chim. Ital. 84,806-815).

A mixture of 0.23 g of 4 and 86 mg of L-tartaric acid (Aldrich) isheated on an oil bath under reduced pressure until fusion at 115° C. Thereaction temperature is increased to 135° C. for 10 min, and thereaction is allowed to cool. The solid product is heated with 4.5 mL of80% acetic acid on a steam bath until dissolved, and then rotovapped.The residue is heated at 50° C. with 1.25 mL of H₂O, and the mixturefiltered to remove unreacted caffeic acid. The filtrate is extracted 2×with ether, and the ether layer is rotovapped. The residue is taken upinto H₂O with warming and adjusted to pH 6 with sodium bicarbonate.Caffeic acid is precipitated as a barium salt by the addition ofsaturated BaSO₄, collected and washed with 3% BaSO₄ bymicrocentrifugation, and then mixed with 0.75 mL 2 M HCl and 2 mL etheruntil in solution. The ether layer is removed and the aqueous phaseextracted 2× with ether. The combined ether extracts are dried overMgSO₄, rotovapped, and the product recrystallized from H₂O to yieldL-chicoric acid (Scarpatti and Oriente, 1958).

Symmetric Anhydride of Bromoacetic (Chloroacetic) Acid

FIG. 3, Scheme 2.

A pre-cooled 0.5 M solution of DCC in DCM (40 ml, 20 mmol) is added to astirred solution of bromoacetic acid (40 mmol) in DCM (20 ml) at 0° C.The reaction mixture is stirred for 30 min and filtered to remove thedicyclohexylurea that have formed, and the filtrate is evaporated on arotary evaporator at 20° C. (Bioconjugate Chemistry 1995, 6, 269).

N-bromoacetyl-N′-Boc-ethylenediamine (3)

FIG. 3, Scheme 2.

Freshly prepared bromoacetic anhydride (20 mmol) is dissolved in 10 mlof acetonitrile, and the solution is added to a stirred solution ofN-Boc-ethylenediamine (18 mmol, Aldrich) and TEA (20 mmol) in THF (20ml) at 20° C. The progress of the reaction if followed by the ninhydrintest for free amines. When all Boc-ethylenediamine is consumed thereaction mixture is concentrated on a rotovap and dissolved in ethylacetate (150 ml). The solution is successively washed with 0.5 M sodiumbicarbonate (50 ml×2), 0.1 M sulfuric acid (50 ml×3), brine (50 ml×2),dried over sodium sulfate and concentrated providing the desiredN-bromoacetyl-N′-Boc-ethylenediamine (3)

N-bromoacetyl-ethylenediamine (4)

FIG. 3, Scheme 2.

N-bromoacetyl-N′-BOC-ethylenediamine (3) is dissolved in 50% TFA indichloromethane (5 ml of the solution per mmol of 3) at 20° C. Thedeprotection is allowed to proceed for 30 min, then the reaction mixtureis concentrated on a rotary evaporator and solidifies upon addition ofdry ethyl ether. The solid material is filtered off, washed withether/petroleum ether on filter and dried. The desiredN-bromoacetyl-ethylenediamine is obtained in the form oftriflouroacetate salt.

Bromoacetylated Derivative of L-Chicoric Acid (6)

FIG. 4, Scheme 3.

Mono NHS ester of L-chicoric acid (5, 0.1 mmol) is added to an excess ofthe N-bromoacetyl-ethylenediamine (0.2 mmol) in a small volume of THF inpresence of DIEA (0.1 mmol). When all the activated ester is consumed,the reaction mixture is diluted with ethyl acetate (150 ml),successively washed with 0.1 M sulfuric acid (100 ml×2) to remove theunreacted amine, brine (50 ml×2), dried over sodium sulfate andevaporated on a rotovap. 6 is further purified by crystallization fromappropriate solvents or by preparative RP HPLC.

Conjugation of 6 with the Recognition/Linkers;

FIG. 5, Scheme 4.

The recognition/linkers from solid phase synthesis (50 μmol) and 6 (60μmol) are dissolved in a small volume of 50 mM sodium acetate buffer, pH4.0 and purged with nitrogen. pH of the solution is raised up to 7-8 byaddition of solid sodium bicarbonate. The reaction mixture is stirred at20° C. until the Elman test shows absence of free thiols in the mixture.The reaction mixture is diluted with 0.1% trifluoroacetic acid and thedesired product is isolated by preparative RP HPLC (Ivanov, B., Grzesik,W., and Robey, F. A. (1995) Bioconjugate Chem. 6, 269-277).

In Vitro Reticulocyte Extract Assay for Targeted Degradation

Degradation is monitored using ¹²⁵I-labeled IN in a rabbit reticulocytelysates by SDS-PAGE/auto radiography and by determination of soluble¹²⁵I after the precipitation of proteins with TCA. The use of thissystem to assess ubiquitin-dependent proteolysis is straightforward andwell established in the literature (Gonda, D. K., Bachmair, A., Wunning,I., Tobias, J. W., Lane, W. S. and Varshavsky, A. (1989) J. Biol. Chem.264, 16700-16712, Hershko, A., Ciechanover, A., Heller, H., Haas, A. L.,and Rose, I. A. (1980) Proc. Natl. Acad. Sci. USA 77, 1783-1786).

The series of trans-targeting compounds are evaluated for their abilityto initiate the degradation of [¹²⁵I]-IN in the reticulocyte lysates.SDS-PAGE time course results show transitory multiubiquitinated INspecies, followed by loss of ¹²⁵I-labeled protein. The assay forTCA-soluble peptide product fragments is used to better quantitate ratesof degradation and effective concentrations.

Preparation of Rabbit Reticulocyte Lysates

Biocon, Inc. (Rockville, Md.) performed the induction and collection ofreticulocytes from NZW rabbits. A female NZW rabbit weighing less than 2kg was injected sub-cutaneous with 0.6 mL/kg of 20 mg/mL phenylhydrazineon day 1,2,4 and 6. On day 8 the rabbit was anesthetized with ketamineand bled out by heart bleed. The blood was collected into heparinizedtubes on ice, and washed 3 times with 5 pellet volumes per wash of coldPBS. The reticulocyte lysates were prepared by the addition of 1.5volumes of cold H₂O 1 mM DTT per volume of packed cells, followed bycentrifugation for 2.5 hrs at 38,400× g. The supernatant was frozen inaliquots at −80° C. The reticulocyte lysate can be used for 2 or 3freeze/thaw cycles only.

TCA Precipitation Assay of [¹²⁵I]-Protein Degradation in ReticulocyteLysates

Proteins were labeled by Lofstrand Laboratories Ltd. (Gaithersburg, Md.)Labeled to 0.12-0.50 μCi/g by oxidation of Na¹²⁵I using an iodobeadchloramine-T procedure. The ubiquitin-dependent protein degradationassay was preformed by the addition of 70 μL of rabbit reticulocytelysate and 5 uL of 0.06 μCi/μL ²⁵I-labeled protein to 175 mL of reactionbuffer containing 40 mM Tris pH 7.6, 2 mM DTT, 5 mM MgCl₂, 0.5 mM ATP,35 μg creatine phosphokinase (Sigma) and 10 mM phosphocreatine. Thereaction were run at 37 C. in a heating block, and at time points 30 μLof the reaction was transferred to 50 μl of cold 100 mg/mL BSA andprotein was precipitated by the addition of 420 μL of 23% TCA followedby 15 min on ice. The precipitated samples were microfuged for 2 min at5000 rpm and 300 μL of supernatant was then counted for TCA-soluble ¹²⁵Ion a gamma counter (Hidex).

Results for [¹²⁵I]-lysozyme(hen, Sigma), [¹²⁵I]-glutathioneS-transferase Degradation

The N-terminal sequences for lysozyme and GST samples submitted toMidwest Analytical were KVFGR and PPYTI, respectively. The only N-endrule stabilizing residues in mammalian cells are Gly, Val, Pro, and Met.Lysozyme is the usual positive control for the reticulocyte lysateassays; GST should be stable to N-end rule, ubiquitin-dependentproteolysis. Time points were taken every 30 min from 0 to 120 min. 25μL r×n samples counted directly in the gamma counter gave 55746 cpm forlysozyme and 45989 cpm or GST. Results demonstrated that the assay wasfunctional for the specific N-end rule degradation as described in theliterature.

TABLE 1 Time course of lysozyme and glutathione S-transferase ubiquitinmediated degradation in the reticulocyte lysate Time Lysozyme GST 0 13381320 30 7512 1474 60 11979 1723 90 14976 1863 120 16337 2173

SDS-PAGE ¹²⁵I protein degradation assay

The ubiquitin-dependent protein degradation assay was preformed by theaddition of 70 μL of rabbit reticulocyte (or other cell) lysate and 5 μLof 0.06 μCi/μL ¹²⁵I-labeled protein to 175 mL of reaction buffercontaining 40 mM Tris pH 7.6, 2 mM DTT, 5 mM MgCl₂, 0.5 mM ATP, 35 μgcreatine phosphokinase (Sigma) and 10 mM phosphocreatine. The reactionwas run at 37 C in a heating block, and at time points 30 μL of thereaction are transferred to gel loading buffer. Samples are run ontricine 10-20% SDS-PAGE gels (Novex) for autoradiography on X-omat film(Kodak) to determine ¹²⁵I protein degradation.

Example 2

Selection, Discovery and/or Evaluation of Ubiquitination RecognitionElements

In order to determine if a given molecule or molecular element ispotentially valuable as a ubiquitination recognition element the assaydescribed above is run with [¹²⁵I]-lysozyme or other labeled proteinsubstrates in the presence of potential ubiquitination recognitionelements.

In the case of Arg-εAhx-Cys, Phe-εAhx-Cys these were run in thereticulocyte lysate using lysozyme and both demonstrated inhibition ofthe lysozyme degradation as expected for ubiquitination recognitionelements. The results at the 2 hour time point were 12,475 cpm for notreatment, 6,486 cpm for the 2 mM Arg-εAhx-Cys treatment and 3,592 cpmfor the 5 mM Phe-εAhx-Cys treatment. These results indicate that aubiquitination recognition element can be made from X-εAhx-linker whereX is an amino acid involved in the N-end recognition and the linker ischemistry which links this to a binding molecule for the target proteinof interest.

In an additional assay for ubiquitination recognition elements compoundsand peptides are added to HeLa or Jurkat cell extracts (Alkalay et al1995, Proc. Natl. Acad. USA 92, 10599), containing radiolabeled IkappaBalpha or IkappaB beta, modulation of the ubiquitination was monitored bygel electrophoresis of the labeled proteins. This allows the selectionof ubiquitination recognition elements specific for the ubiquitinationpathway used for IkappaB degradation (Yaron A, 1997, EMBO J. 16, 6486).

Example 3

Targeted Degradation of Glutathione S-transferase

Conjugation of Ubiquitination Recognition Elements to Glutathione (FIG.6)

4.18 mg (15.1 μmol) bismaleimidohexane (BMH, Pierce, Rockford, Ill.) in200 μL of dimethylformamide was added slowly to 1.84 mg (6 μmol)glutathione in 2 mL 20 mM potassium phosphate pH 7.0 The reaction wasfollowed by C₁₈ reverse phase HPLC. After 30 min at room temperature,the reaction mixture was centrifuged at 12,000 rpm for 2 min to removeprecipitate, and the sample was loaded onto a C₁₈ Sep-Pak cartridgepre-equilibrated with H₂O. The bound sample was washed with 2 mL of 10%methanol/H₂O and eluted in 3 1 -mL fractions of 50% methanol. The second1 mL product fraction was partially concentrated by evaporation of themethanol. This activated glutathione was then reacted with the variousubiquitination recognition elements.

For example for Arg-εAhx-Cys the activated glutathione was added to 50μL of 20 mg/mL Arg-εAhx-Cys. The pH was adjusted to pH 6.5 by theaddition of 5 M sodium hydroxide and the reaction was followed by C₁₈reverse phase HPLC. This protocol repeated for the followingubiquitination recognition elements, Arg-εAhx-Cys, Arg-β-Ala-εAhx-Cys,Arg-εAhx-εAhx-Cys, Phe-εAhx-Cys, Phe-β-Ala-εAhx-Cys, Phe-εAhx-εAhx-Cys,KKERLLDDRHDSGLDSMKDEEC (SEQ ID NO 50) where the S in bold arephosphorylated, RAALAVLKSGNC (SEQ ID NO 51),HGFPPEVEEQDVGTLPISCAQESGMDRHC (SEQ ID NO 52). This generated a series ofcompounds for testing in the rabbit reticulocyte, HeLa cell and Jurkatcell lysates.

Glutathione S-transferase (Sigma, St. Louis, Mo.) was labeled byLofstrand Laboratories Ltd. (Gaithersburg, Md.) Labeled to 0.12-0.50μCi/μg by oxidation of Na¹²⁵I using an iodobead chloramine-T procedure.

The ubiquitin-dependent protein degradation assay was preformed by theaddition of 70 μL of rabbit reticulocyte (or other cell) lysate and 5 μLof 0.06 μCi/μL ¹²⁵I-labeled Glutathione S-transferase to 175 mL ofreaction buffer containing 40 mM Tris pH 7.6, 2 mM DTT, 5 mM MgCl₂, 0.5mM ATP, 35 μg creatine phosphokinase (Sigma) and 10 mM phosphocreatine.To demonstrate the targeted degradation of the GST, variousconcentrations of the compounds from the above synthesis were added tothe lysate (10 to 0.001 mM). The reaction were run at 37 C. in a heatingblock, and at time points 30 μL of the reaction was transferred to 50 μLof cold 100 mg/mL BSA and protein was precipitated by the addition of420 μL of 23% TCA followed by 15 min on ice. The precipitated sampleswere microfuged for 2 min at 5000 rpm and 300 μL of supernatant was thencounted for TCA-soluble ¹²⁵I on a gamma counter (Hidex). Time points aretaken every 30 min from 0 to 120 min. Results demonstrate targeteddegradation.

Example 4

Targeted Degradation of Anti Fluorescein Antibody

Conjugation of Ubiquitination Recognition Elements toFluorescein-5-maleimide (FIG. 7)

Arg-εAhx-Cys. To 400 μL of 5 mg/mL Arg-εAhx-Cys (2.00 mg, 5.13 μmol) wasadded 219 μL of 50 mg/mL fluorescein-5-maleimide in dimethylformamide(Pierce, 10.95 mg, 25.65 μmol, 5-fold molar excess) and the pH wasadjusted to pH 6.5 with 5 M sodium hydroxide. The reaction was followedby C₁₈ reverse phase HPLC. After 60 min at room temperature, the samplewas loaded onto a C₁₈ Sep-Pak cartridge pre-equilibrated with H₂O. Thebound sample was washed with 2 mL of 10% methanol/H₂O and the producteluted in 3 1 mL fractions of 60% methanol. This protocol is repeatedfor the following ubiquitination recognition elements, Arg-εAhx-Cys,Arg-β-Ala-εAhx-Cys, Arg-εAhx-εAhx-Cys, Phe-εAhx-Cys, Phe-β-Ala-εAhx-Cys,Phe-εAhx-εAhx-Cys. KKERLLDDRHDSGLDSMKDEEC (SEQ ID NO 50) where the S inbold are phosphorylated, RAALAVLKSGNC (SEQ ID NO 51),HGFPPEVEEQDVGTLPISCAQESGMDRHC (SEQ ID NO 52). This generated a series ofcompounds for testing in the rabbit reticulocyte lysate.

Anti fluorescein antibodies (Fitzgerald and Molecular Probes, OR) waslabeled by Lofstrand Laboratories Ltd. (Gaithersburg, Md.) Labeled to0.12-0.50 μCi/μg by oxidation of Na¹²⁵I using an iodobead chloramine-Tprocedure.

The ubiquitin-dependent protein degradation assay was preformed by theaddition of 70 μL of rabbit reticulocyte (or other cell) lysate and 5 μLof 0.06 μCi/μL ¹²⁵I-labeled anti fluorescein antibody to 175 mL ofreaction buffer containing 40 mM Tris pH 7.6, 2 mM DTT, 5 mM MgCl₂, 0.5mM ATP, 35 μg creatine phosphokinase (Sigma) and 10 mM phosphocreatine.To demonstrate the targeted degradation of the anti fluoresceinantibodies, various concentrations of the compounds from the abovesynthesis were added to the lysate (10 to 0.001 mM). The reaction wererun at 37 C in a heating block, and at time points 30 μL of the reactionwas transferred to 50 μL of cold 100 mg/mL BSA and protein wasprecipitated by the addition of 420 μL of 23% TCA followed by 15 min onice. The precipitated samples were microfuged for 2 min at 5000 rpm and300 μL of supernatant was then counted for TCA-soluble ¹²⁵I on a gammacounter (Hidex). Time points are taken every 30 min from 0 to 120 min.Results demonstrate the targeted degradation.

Example 5

Targeted Degradation of Thioredoxin

Conjugation of Ubiquitination Recognition Elements to 4-aminophenylArsenoxide.

Arg-εAhx-Cys. To 1.83 mg (10 μmol) 4-aminophenyl arsenoxide in 100 μLdimethylformamide was added 5.3 μL of 50 mg/mL ethyleneglycobis(sulfo-succinimidylsuccinate) (Pierce, 15 μmol, 1.5 equivalents)in dimethyformamide. After 30 min reaction time at room temperature, 2.0mg (5.13 μmol) Arg-εAhx-Lys in 400 μL 20 mM potassium phosphate pH 6.5.The reaction was followed by C₁₈ reverse phase HPLC. After 30 min, thederivatized peptide product was separated from the reaction mixture byC₁₈ Sep-Pak solid phase extraction. The bound sample was washed with 2mL of 10% methaol/H₂O and eluted in 3 1 mL fractions of 60% methanol.This protocol is repeated for the following ubiquitination recognitionelements, Arg-εAhx-Lys, Arg-β-Ala-εAhx-Lys, Arg-εAhx-εAhx-Lys,Phe-εAhx-Lys, Phe-β-Ala-εAhx-Lys, Phe-εAhx-εAhx-Lys,KAADADEWCDSGLGSLGPDA (SEQ ID NO 42) where the S in bold arephosphorylated, RHALDDVSNK (SEQ ID NO 54), HGFPPEVEEQDVGTLPISCAQESGMDRHK(SEQ ID NO 55). This generated a series of compounds for testing in therabbit reticulocyte lysate.

Thioredoxin was prepared following standard method from the plasmidvector pBAD/Thio, (Invitrogen, Carlsbad, Calif.). The plasmid vector wastransformed into TOP10 cells and colonies grown up in LB with 50micrograms/ml ampicillin overnight at 37 C. This overnight culture wasthen used to inoculate a large culture of LB with 50 micrograms/mlampicillin and supplemented with arabinose to induce expression. Theculture was then harvested and lysed by sonication and run on to aProBond™ column (Invitrogen), following the manufactures protocol toyield purified thioredoxin.

Thioredoxin was labeled by Lofstrand Laboratories Ltd. (Gaithersburg,Md.) Labeled to 0.12-0.50 μCi/μg by oxidation of Na¹²⁵I using aniodobead chloramine-T procedure.

The ubiquitin-dependent protein degradation assay was preformed by theaddition of 70 μL of rabbit reticulocyte lysate and 5 μL of 0.06 μCi/μL¹²⁵I-labeled thioredoxin to 175 mL of reaction buffer containing 40 mMTris pH 7.6, 2 mM DTT, 5 mM MgCl₂, 0.5 mM ATP, 35 μg creatinephosphokinase (Sigma) and 10 mM phosphocreatine. To demonstrate thetargeted degradation of the thioredoxin, various concentrations of thecompounds from the above synthesis were added to the lysate (10 to 0.001mM). The reaction were run at 37 C. in a heating block, and at timepoints 30 μL of the reaction was transferred to 50 μL of cold 100 mg/mLBSA and protein was precipitated by the addition of 420 μL of 23% TCAfollowed by 15 min on ice. The precipitated samples were microfuged for2 min at 5000 rpm and 300 μL of supernatant was then counted forTCA-soluble ¹²⁵I on a gamma counter (Hidex).

Time points are taken every 30 min from 0 to 120 min. Resultsdemonstrate the targeted degradation.

It will be readily apparent to those skilled in the art that numerousmodifications and additions may be made to both the present inventionwithout departing from the invention disclosed.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 67 <210> SEQ ID NO 1 <211> LENGTH: 20<212> TYPE: PRT <213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: PEST example      sequence <400> SEQUENCE: 1Met Glu Phe Met His Ile Ser Pro Pro Glu Pr #o Glu Ser Glu Glu Glu  1               5  #                 10  #                 15Glu Glu His Ser              20 <210> SEQ ID NO 2 <211> LENGTH: 10<212> TYPE: PRT <213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: PEST example      sequence <400> SEQUENCE: 2 Met Glu Phe Met His Glu Ser His Ser Ser  1               5  #                 10 <210> SEQ ID NO 3<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Unknown Organism<220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Or#ganism: PEST example       sequence <400> SEQUENCE: 3Met Glu Phe Met His Ile Ser Pro Pro Glu Pr #o Glu Ser His Ser Ser  1               5  #                 10  #                 15<210> SEQ ID NO 4 <211> LENGTH: 15 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: PEST example      sequence <400> SEQUENCE: 4Met Glu Phe Met His Glu Ser Glu Glu Glu Gl #u Glu His Ser Ser  1               5  #                 10  #                 15<210> SEQ ID NO 5 <211> LENGTH: 10 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: PEST example      sequence <400> SEQUENCE: 5 Met Glu Ala Ser Glu Glu Glu Glu Glu Phe  1               5  #                 10 <210> SEQ ID NO 6<211> LENGTH: 28 <212> TYPE: PRT <213> ORGANISM: Unknown Organism<220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Or#ganism: PEST example       sequence <400> SEQUENCE: 6His Gly Phe Pro Pro Glu Val Glu Glu Gln As #p Asp Gly Thr Leu Pro  1               5  #                 10  #                 15Met Ser Cys Ala Gln Glu Ser Gly Met Asp Ar #g His              20     #             25 <210> SEQ ID NO 7 <211> LENGTH: 28 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: PEST example      sequence <400> SEQUENCE: 7His Gly Phe Pro Pro Ala Val Ala Ala Gln As #p Asp Gly Thr Leu Pro  1               5  #                 10  #                 15Met Ser Cys Ala Gln Glu Ser Gly Met Asp Ar #g His              20     #             25 <210> SEQ ID NO 8 <211> LENGTH: 28 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: PEST example      sequence <400> SEQUENCE: 8His Gly Phe Pro Pro Glu Val Glu Glu Gln As #p Asp Gly Ala Leu Pro  1               5  #                 10  #                 15Met Ser Cys Ala Gln Glu Ser Gly Met Asp Ar #g His              20     #             25 <210> SEQ ID NO 9 <211> LENGTH: 28 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: PEST example      sequence <400> SEQUENCE: 9His Gly Phe Pro Pro Glu Val Glu Glu Gln As #p Asp Gly Thr Leu Pro  1               5  #                 10  #                 15Met Ser Cys Ala Gln Glu Ser Gly Met Asp Hi #s His              20     #             25 <210> SEQ ID NO 10 <211> LENGTH: 28 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: PEST example      sequence <400> SEQUENCE: 10His Gly Phe Pro Pro Glu Val Glu Glu Gln As #p Val Gly Thr Leu Pro  1               5  #                 10  #                 15Met Ser Cys Ala Gln Glu Ser Gly Met Asp Ar #g His              20     #             25 <210> SEQ ID NO 11 <211> LENGTH: 28 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: PEST example      sequence <400> SEQUENCE: 11His Gly Phe Pro Pro Glu Val Glu Glu Gln As #p Val Gly Thr Leu Pro  1               5  #                 10  #                 15Ile Ser Cys Ala Gln Glu Ser Gly Met Asp Ar #g His              20     #             25 <210> SEQ ID NO 12 <211> LENGTH: 28 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: PEST example      sequence <400> SEQUENCE: 12His Gly Phe Pro Pro Glu Val Glu Glu Gln As #p Ala Ser Thr Leu Pro  1               5  #                 10  #                 15Val Ser Cys Ala Trp Glu Ser Gly Met Lys Ar #g His              20     #             25 <210> SEQ ID NO 13 <211> LENGTH: 26 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: PEST example      sequence <400> SEQUENCE: 13Phe Pro Pro Gly Val Glu Glu Pro Asp Val Gl #y Pro Leu Pro Val Ser  1               5  #                 10  #                 15Cys Ala Trp Glu Ser Gly Met Lys Arg His              20     #             25 <210> SEQ ID NO 14 <211> LENGTH: 27 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: PEST example      sequence <400> SEQUENCE: 14Phe Leu Ala Glu Val Glu Glu Gln Asp Val Al #a Ser Leu Pro Leu Ser  1               5  #                 10  #                 15Cys Ala Cys Glu Ser Gly Ile Glu Tyr Pro Al #a              20     #             25 <210> SEQ ID NO 15 <211> LENGTH: 25 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: consensus      sequence <221> NAME/KEY: MOD_RES <222> LOCATION: (2)..(3)<223> OTHER INFORMATION: any amino acid <221> NAME/KEY: MOD_RES<222> LOCATION: (10)..(12) <223> OTHER INFORMATION: any amino acid<221> NAME/KEY: MOD_RES <222> LOCATION: (15)<223> OTHER INFORMATION: any amino acid <221> NAME/KEY: MOD_RES<222> LOCATION: (19) <223> OTHER INFORMATION: any amino acid<221> NAME/KEY: MOD_RES <222> LOCATION: (23)..(24)<223> OTHER INFORMATION: any amino acid <221> NAME/KEY: MOD_RES<222> LOCATION: (25) <223> OTHER INFORMATION: optional amino acid<400> SEQUENCE: 15 Phe Xaa Xaa Glu Val Glu Glu Gln Asp Xaa Xa#a Xaa Leu Pro Xaa Ser   1               5  #                 10 #                 15 Cys Ala Xaa Glu Ser Gly Xaa Xaa Xaa             20      #             25 <210> SEQ ID NO 16<211> LENGTH: 26 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: consensus       sequence <221> NAME/KEY: MOD_RES<222> LOCATION: (2)..(3) <223> OTHER INFORMATION: any amino acid<221> NAME/KEY: MOD_RES <222> LOCATION: (10)..(12)<223> OTHER INFORMATION: any amino acid <221> NAME/KEY: MOD_RES<222> LOCATION: (15) <223> OTHER INFORMATION: any amino acid<221> NAME/KEY: MOD_RES <222> LOCATION: (19)<223> OTHER INFORMATION: any amino acid <221> NAME/KEY: MOD_RES<222> LOCATION: (23)..(24) <223> OTHER INFORMATION: any amino acid<221> NAME/KEY: MOD_RES <222> LOCATION: (25)<223> OTHER INFORMATION: optional amino acid <221> NAME/KEY: MOD_RES<222> LOCATION: (26) <223> OTHER INFORMATION: any amino acid<400> SEQUENCE: 16 Phe Xaa Xaa Ala Val Ala Ala Gln Asp Xaa Xa#a Xaa Leu Pro Xaa Ser   1               5  #                 10 #                 15 Cys Ala Xaa Glu Ser Gly Xaa Xaa Xaa Xaa             20      #             25 <210> SEQ ID NO 17<211> LENGTH: 28 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: consensus       sequence <221> NAME/KEY: MOD_RES<222> LOCATION: (3)..(4) <223> OTHER INFORMATION: any amino acid<221> NAME/KEY: MOD_RES <222> LOCATION: (8)<223> OTHER INFORMATION: any amino acid <221> NAME/KEY: MOD_RES<222> LOCATION: (9)..(10) <223> OTHER INFORMATION: optional amino acid<221> NAME/KEY: MOD_RES <222> LOCATION: (12)..(14)<223> OTHER INFORMATION: any amino acid <221> NAME/KEY: MOD_RES<222> LOCATION: (16)..(17) <223> OTHER INFORMATION: any amino acid<221> NAME/KEY: MOD_RES <222> LOCATION: (26)..(28)<223> OTHER INFORMATION: any amino acid <400> SEQUENCE: 17His Gly Xaa Xaa Pro Glu Val Xaa Xaa Xaa As #p Xaa Xaa Xaa Leu Xaa  1               5  #                 10  #                 15Xaa Ser Cys Ala Gln Glu Ser Gly Met Xaa Xa #a Xaa              20     #             25 <210> SEQ ID NO 18 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 18Arg His Ala Leu Asp Asp Val Ser Asn   1               5<210> SEQ ID NO 19 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 19Arg Leu Ala Leu Asn Asn Val Thr Asn   1               5<210> SEQ ID NO 20 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 20Arg Ala Ala Leu Gly Asp Val Ser Asn   1               5<210> SEQ ID NO 21 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 21Arg Gln Val Leu Gly Asp Ile Gly Asn   1               5<210> SEQ ID NO 22 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 22Arg Ala Ala Leu Gly Asp Leu Gln Asn   1               5<210> SEQ ID NO 23 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 23Arg Ala Ala Leu Gly Asn Ile Ser Asn   1               5<210> SEQ ID NO 24 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 24Arg Asn Thr Leu Gly Asp Ile Gly Asn   1               5<210> SEQ ID NO 25 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 25Arg Thr Ala Leu Gly Asp Ile Gly Asn   1               5<210> SEQ ID NO 26 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 26Arg Ala Ala Leu Gly Glu Ile Gly Asn   1               5<210> SEQ ID NO 27 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 27Arg Ala Val Leu Glu Glu Ile Gly Asn   1               5<210> SEQ ID NO 28 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 28Arg Ser Ala Phe Gly Asp Ile Thr Asn   1               5<210> SEQ ID NO 29 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 29Arg Ser Ile Leu Gly Val Ile Gln Ser   1               5<210> SEQ ID NO 30 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 30Arg Ala Ala Leu Gly Val Ile Thr Asn   1               5<210> SEQ ID NO 31 <211> LENGTH: 10 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 31Arg Thr Val Leu Gly Val Ile Gly Asp Asn   1               5 #                 10 <210> SEQ ID NO 32 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 32Arg Thr Val Gly Val Leu Gln Glu Asn   1               5<210> SEQ ID NO 33 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 33Arg Ala Ala Leu Gly Thr Val Gly Glu   1               5<210> SEQ ID NO 34 <211> LENGTH: 10 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 34Arg Thr Val Leu Gly Val Leu Thr Glu Asn   1               5 #                 10 <210> SEQ ID NO 35 <211> LENGTH: 11 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 35Arg Ala Ala Leu Ala Val Leu Lys Ser Gly As #n   1               5 #                 10 <210> SEQ ID NO 36 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 36Arg Leu Pro Leu Ala Ala Lys Asp Asn   1               5<210> SEQ ID NO 37 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 37Arg Gln Leu Phe Pro Ile Pro Leu Asn   1               5<210> SEQ ID NO 38 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box example       sequence <400> SEQUENCE: 38Arg Arg Thr Leu Lys Val Ile Gln Pro   1               5<210> SEQ ID NO 39 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: D box general       structure <221> NAME/KEY: MOD_RES<222> LOCATION: (2) <223> OTHER INFORMATION: Ala or Thr<221> NAME/KEY: MOD_RES <222> LOCATION: (3)<223> OTHER INFORMATION: amino acid present more  #than %50 of the time<221> NAME/KEY: MOD_RES <222> LOCATION: (6)<223> OTHER INFORMATION: any amino acid <221> NAME/KEY: MOD_RES<222> LOCATION: (7) <223> OTHER INFORMATION: Ile or Val<221> NAME/KEY: MOD_RES <222> LOCATION: (8)<223> OTHER INFORMATION: Gly or Thr <221> NAME/KEY: MOD_RES<222> LOCATION: (9) <223> OTHER INFORMATION: amino acid present more #than %50 of the time <400> SEQUENCE: 39Arg Xaa Ala Leu Gly Xaa Xaa Xaa Asn   1               5<210> SEQ ID NO 40 <211> LENGTH: 21 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: ubiquitination       recognition element <400> SEQUENCE: 40Lys Glu Phe Ala Val Pro Asn Glu Thr Ser As #p Ser Gly Phe Ile Ser  1               5  #                 10  #                 15Gly Pro Gln Ser Ser              20 <210> SEQ ID NO 41 <211> LENGTH: 22<212> TYPE: PRT <213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: ubiquitination       recognition element <400> SEQUENCE: 41Lys Gly Pro Asp Glu Ala Glu Glu Ser Gln Ty #r Asp Ser Gly Leu Glu  1               5  #                 10  #                 15Ser Leu Arg Ser Leu Arg              20 <210> SEQ ID NO 42<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Unknown Organism<220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Or#ganism: ubiquitination       recognition element <400> SEQUENCE: 42Lys Ala Ala Asp Ala Asp Glu Trp Cys Asp Se #r Gly Leu Gly Ser Leu  1               5  #                 10  #                 15Gly Pro Asp Ala              20 <210> SEQ ID NO 43 <211> LENGTH: 21<212> TYPE: PRT <213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: ubiquitination       recognition element <400> SEQUENCE: 43Lys Lys Glu Arg Leu Leu Asp Asp Arg His As #p Ser Gly Leu Asp Ser  1               5  #                 10  #                 15Met Lys Asp Glu Glu              20 <210> SEQ ID NO 44 <211> LENGTH: 14<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: consensus      sequence <221> NAME/KEY: MOD_RES <222> LOCATION: (2)...(11)<223> OTHER INFORMATION: any amino acid<223> OTHER INFORMATION: positions 2-11 may encomp #ass X(8-10)<400> SEQUENCE: 44 Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa#a Asp Ser Gly   1               5  #                 10<210> SEQ ID NO 45 <211> LENGTH: 12 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: ubiqitination       recognition element <400> SEQUENCE: 45Ser Tyr Leu Asp Ser Gly Ile His Ser Gly Al #a Thr   1               5 #                 10 <210> SEQ ID NO 46 <211> LENGTH: 12 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: ubiqitination       recognition element <400> SEQUENCE: 46Arg Ala Glu Asp Ser Gly Asn Glu Ser Glu Gl #y Glu   1               5 #                 10 <210> SEQ ID NO 47 <211> LENGTH: 6 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: example      peptide <221> NAME/KEY: MOD_RES <222> LOCATION: (3)...(4)<223> OTHER INFORMATION: any amino acid <400> SEQUENCE: 47Cys Cys Xaa Xaa Cys Cys   1               5 <210> SEQ ID NO 48<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Unknown Organism<220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Or#ganism: example       peptide <400> SEQUENCE: 48Trp Glu Ala Ala Ala Arg Glu Ala Cys Cys Ar #g Glu Cys Cys Ala Arg  1               5  #                 10  #                 15 Ala<210> SEQ ID NO 49 <211> LENGTH: 17 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: example      peptide <400> SEQUENCE: 49Ala Glu Ala Ala Ala Arg Glu Ala Cys Cys Ar #g Glu Cys Cys Ala Arg  1               5  #                 10  #                 15 Ala<210> SEQ ID NO 50 <211> LENGTH: 22 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: ubiquitination       recognition element <400> SEQUENCE: 50Lys Lys Glu Arg Leu Leu Asp Asp Arg His As #p Ser Gly Leu Asp Ser  1               5  #                 10  #                 15Met Lys Asp Glu Glu Cys              20 <210> SEQ ID NO 51<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Unknown Organism<220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Or#ganism: ubiquitination       recognition element <400> SEQUENCE: 51Arg Ala Ala Leu Ala Val Leu Lys Ser Gly As #n Cys   1               5 #                 10 <210> SEQ ID NO 52 <211> LENGTH: 29 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: ubiquitination       recognition element <400> SEQUENCE: 52His Gly Phe Pro Pro Glu Val Glu Glu Gln As #p Val Gly Thr Leu Pro  1               5  #                 10  #                 15Ile Ser Cys Ala Gln Glu Ser Gly Met Asp Ar #g His Cys             20      #             25 <210> SEQ ID NO 53 <211> LENGTH: 9<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: consensus      sequence <221> NAME/KEY: MOD_RES <222> LOCATION: (2)...(3)<223> OTHER INFORMATION: any amino acid <221> NAME/KEY: MOD_RES<222> LOCATION: (6) <223> OTHER INFORMATION: any amino acid<221> NAME/KEY: MOD_RES <222> LOCATION: (8)<223> OTHER INFORMATION: any amino acid <400> SEQUENCE: 53Arg Xaa Xaa Leu Gly Xaa Ile Xaa Asn   1               5<210> SEQ ID NO 54 <211> LENGTH: 10 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: ubiquitination       recognition element <400> SEQUENCE: 54Arg His Ala Leu Asp Asp Val Ser Asn Lys   1               5 #                 10 <210> SEQ ID NO 55 <211> LENGTH: 29 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: ubiquitination       recognition element <400> SEQUENCE: 55His Gly Phe Pro Pro Glu Val Glu Glu Gln As #p Val Gly Thr Leu Pro  1               5  #                 10  #                 15Ile Ser Cys Ala Gln Glu Ser Gly Met Asp Ar #g His Lys             20      #             25 <210> SEQ ID NO 56 <211> LENGTH: 4<212> TYPE: PRT <213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: binding      peptide <400> SEQUENCE: 56 Tyr Glu Glu Ile   1 <210> SEQ ID NO 57<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Unknown Organism<220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Or#ganism: binding       peptide <400> SEQUENCE: 57Asp Arg Glu Gly Cys Arg Arg Gly Trp Val Gl #y Gln Cys Lys Ala Trp  1               5  #                 10  #                 15 Phe Asn<210> SEQ ID NO 58 <211> LENGTH: 22 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: binding      peptide <400> SEQUENCE: 58Glu Thr Pro Thr Phe Thr Trp Glu Glu Ser As #n Ala Tyr Tyr Trp Gln  1               5  #                 10  #                 15Pro Tyr Ala Leu Pro Leu              20 <210> SEQ ID NO 59<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Unknown Organism<220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Or#ganism: binding       peptide <400> SEQUENCE: 59Thr Phe Val Tyr Trp Gln Pro Tyr Ala Leu Pr #o Leu   1               5 #                 10 <210> SEQ ID NO 60 <211> LENGTH: 15 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: binding      peptide <400> SEQUENCE: 60Val Ser Leu Ala Arg Arg Pro Leu Pro Pro Le #u Pro Gly Gly Lys  1               5  #                 10  #                 15<210> SEQ ID NO 61 <211> LENGTH: 17 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: binding      peptide <400> SEQUENCE: 61Lys Gly Gly Gly Ala Ala Pro Pro Leu Pro Pr #o Arg Asn Arg Pro Arg  1               5  #                 10  #                 15 Leu<210> SEQ ID NO 62 <211> LENGTH: 15 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: binding      peptide <400> SEQUENCE: 62Ala Glu Cys His Pro Gln Gly Pro Pro Cys Il #e Glu Gly Arg Lys  1               5  #                 10  #                 15<210> SEQ ID NO 63 <211> LENGTH: 13 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: binding      peptide <400> SEQUENCE: 63Gly Ala Cys Arg Arg Glu Thr Ala Trp Ala Cy #s Gly Ala  1               5  #                 10 <210> SEQ ID NO 64<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Unknown Organism<220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Or#ganism: binding       peptide <400> SEQUENCE: 64Asp Ile Thr Trp Asp Gln Leu Trp Asp Leu Me #t Lys   1               5 #                 10 <210> SEQ ID NO 65 <211> LENGTH: 13 <212> TYPE: PRT<213> ORGANISM: Unknown Organism <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: binding      peptide <400> SEQUENCE: 65Arg Asn Met Ser Trp Leu Glu Leu Trp Glu Hi #s Met Lys  1               5  #                 10 <210> SEQ ID NO 66<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Unknown Organism<220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Or#ganism: ubiquitination       recognition element<221> NAME/KEY: MOD_RES <222> LOCATION: (3)<223> OTHER INFORMATION: beta-Ala<223> OTHER INFORMATION: caproic acid linker betwe #en positions 3-4<400> SEQUENCE: 66 Arg Ala Ala Cys   1 <210> SEQ ID NO 67<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Unknown Organism<220> FEATURE: <223> OTHER INFORMATION: Description of Unknown Or#ganism: ubiquitination       recognition element<221> NAME/KEY: MOD_RES <222> LOCATION: (3)<223> OTHER INFORMATION: beta-Ala<223> OTHER INFORMATION: caproic acid linker betwe #en positions 3-4<400> SEQUENCE: 67 Pro Ala Ala Cys   1

What is claimed is:
 1. A compound for activating ubiquitination of atarget protein comprising; a) a ubiquitination recognition element whichis able to bind to either the E3 or E2 functional elements of theubiquitination system, wherein said ubiquitination recognition elementhas a molecular weight less than 30,000 and has a binding affinity forsaid E3 and/or E2 elements of the ubiquitination system of at least 10²M⁻¹, and; b) a target protein binding element that is able to bindspecifically to a target protein wherein said target protein bindingelement has a molecular weight of less than 30,000 and has a bindingaffinity for said target protein greater than 10⁵ M⁻¹, wherein saidubiquitination recognition element is covalently linked to said targetprotein binding element.
 2. A compound for activating ubiquitination ofa target protein comprising; a) a ubiquitination recognition peptideelement which is able to bind to either the E3 or E2 functional elementsof the ubiquitination system, wherein said ubiquitination recognitionpeptide element has a molecular weight less than 30,000 and has abinding affinity for said E3 and/or E2 elements of the ubiquitinationsystem of at least 10² M⁻¹, b) a target protein binding element that isable to bind specifically to a target protein wherein said targetprotein binding element has a molecular weight of less than 30,000 andhas a binding affinity for said target protein greater than 10⁵ M⁻¹,wherein said ubiquitination recognition peptide element is covalentlylinked to said target protein binding element.
 3. A compound foractivating ubiquitination of a target protein comprising; a) aubiquitination recognition element which is able to bind to either theE3 or E2 functional elements of the ubiquitination system, wherein saidubiquitination recognition element has a molecular weight less than30,000 and has a binding affinity for said E3 and/or E2 elements of theubiquitination system of at least 10² M⁻¹ and; b) a target proteinbinding peptide element that is able to bind specifically to a targetprotein wherein said target protein binding peptide element has amolecular weight of less than 30,000 and has a binding affinity for saidtarget protein greater than 10⁵ M⁻¹, wherein said ubiquitinationrecognition element is covalently linked to said target protein bindingpeptide element.
 4. A compound for activating ubiquitination of a targetprotein comprising; a) a ubiquitination recognition peptide elementwhich is able to bind to either the 3 or E2 elements of theubiquitination system, wherein said ubiquitination recognition peptideelement has a molecular weight less than 30,000 and has a bindingaffinity for said E3 and/or E2 elements of the ubiquitination system ofat least 10² M⁻¹ and; b) a target protein binding peptide element thatis able to bind specifically to a target protein wherein said targetprotein binding peptide element has a molecular weight of less than30,000 and has a binding affinity for said target protein greater than10⁵ M⁻¹ wherein said ubiquitination recognition peptide element iscovalently linked to said target protein binding peptide element.
 5. Acompound as in claim 1 wherein said ubiquitination recognition elementhas an affinity of at least 10³ M⁻¹ and a molecular weight between 50and 10,000.
 6. A compound as in claim 5 wherein said target proteinbinding element has a molecular weight from 50 to 10,000 and a bindingaffinity of greater than 10⁶ M⁻¹.
 7. A compound as in claim 1 whereinsaid ubiquitination recognition element has an affinity of at least 10⁴M⁻¹ and a molecular weight between 50 and 3,000.
 8. A compound as inclaim 1 wherein said target protein binding element has a molecularweight from 50 to 3,000 and a binding affinity of greater than 10⁷ M⁻¹.9. A compound as in claim 5 wherein said target protein binding elementhas a molecular weight from 50 to 3,000 and a binding affinity ofgreater than 10⁸ M⁻¹.
 10. A compound as in claim 1 wherein saidubiquitination recognition element contains an amino acid with a freeamino terminal selected from the group consisting of Phe, Arg, Lys, Trp,Leu, Asn, Asp, Gln, Tyr, His, Glu, Cys, Thr, Ser and Ala and oxidizedderivatives thereof.
 11. A compound as in claim 1 wherein saidubiquitination recognition element contains an amino acid selected fromthe group consisting of Phe, Arg, Lys, Asn, Asp, Gln, Glu and Cys.
 12. Acompound as in claim 1 wherein said ubiquitination recognition elementcontains an amino acid selected from the group consisting of Arg, Phe,Asp, Gln and Glu.
 13. A compound as in claim 1 wherein saidubiquitination recognition element contains a moiety selected from thegroup consisting of Arg-εAhx-Cys, Arg-β-Ala-εAhx-Cys, Arg-εAhx-εAhx-Cys,Phe-εAhx-Cys, Phe-β-Ala-εAhx-Cys, Phe-εAhx-εAhx-Cys, Arg-Ala-εAhx-Cys,Arg-Ala-β-Ala-εAhx-Cys (SEQ ID NO:66), Arg-Ala-εAhx-εAhx-Cys,Phe-Ala-εAhx-Cys, Phe-Ala-β-Ala-εAhx-Cys (SEQ ID NO:67) andPhe-Ala-εAhx-εAhx-Cys.
 14. A compound as in claim 1 wherein saidubiquitination recognition element contains a moiety selected from thegroup consisting of; Arg-εAhx-Cys, Arg-β-Ala-εAhx-Cys,Arg-εAhx-εAhx-Cys, Phe-εAhx-Cys, Phe-β-Ala-εAhx-Cys, Phe-εAhx-εAhx-Cys.15. A compound as in claim 1 wherein said recognition element contains amoiety selected from the group consisting of Phe-εAhx-Cys,Phe-β-Ala-εAhx-Cys, Phe-εAhx-εAhx-Cys.
 16. A compound as in claim 1wherein said ubiquitination recognition element is a compound able tointeract with the recognition site of the ubiquitination system, saidrecognition sites selected from the recognition sites for aubiquitination recognition signal selected from the group consisting ofN-end N-recognin, ‘destruction box’ or D box, PEST motifs, Deg1, Deg 2,delta (δ) domains, WW domain binding peptides and phosphorylatedsequences.
 17. A compound as in claim 1 wherein said degradation resultsin altered presentation of degradation products on MHC proteins.
 18. Acompound as in claim 17 wherein said MHC proteins are selected from MHCclass I and MHC class II.
 19. A compound as in claim 1 wherein saidcompound has a molecular weight of less than 3,000.
 20. A compound as inclaim I wherein said ubiquitination recognition element binds the sameubiquitination recognition site as an N-recognin.
 21. A ubiquitinationrecognition element comprising at least one structural element selectedfrom the group consisting of compound Z, Arg-εAhx-linker,Arg-β-Ala-εAhx-linker, Arg-εAhx-εAhx-linker, Phe-εAhx-linker,Phe-β-Ala-εAhx-linker, Phe-εAhx-εAhx-linker, Arg-Ala-εAhx-linker,Arg-Ala-β-Ala-εAhx-linker, Arg-Ala-εAhx-εAhx-linker,Phe-Ala-εAhx-linker, Phe-Ala-β-Ala-εAhx-linker,Phe-Ala-εAhx-εAhx-linker.